Structure-Function Relationships of the α1b-Adrenergic Receptor

The aim of this review is to provide a systematic overview on constitutively active G-protein-coupled receptors (GPCRs), a rapidly evolving area in signal transduction research. We will discuss mechanisms, pharmacological tools and methodological approaches to analyze constitutive activity. The two-state model defines constitutive activity as the ability of a GPCR to undergo agonist-independent isomerization from an inactive (R) state to an active (R*) state. While the two-state model explains basic concepts of constitutive GPCR activity and inverse agonism, there is increasing evidence for multiple active GPCR conformations with distinct biological activities. As a result of constitutive GPCR activity, basal G-protein activity increases. Until now, constitutive activity has been observed for more than 60 wild-type GPCRs from the families 1-3 and from different species including humans and commonly used laboratory animal species. Additionally, several naturally occurring and disease-causing GPCR mutants with increased constitutive activity relative to wild-type GPCRs have been identified. Alternative splicing, RNA editing, polymorphisms within a given species, species variants and coupling to specific G-proteins all modulate the constitutive activity of GPCRs, providing multiple regulatory switches to fine-tune basal cellular activities. The most important pharmacological tools to analyze constitutive activity are inverse agonists and Na(+) that stabilize the R state, and pertussis toxin that uncouples GPCRs from G(i)/G(o)-proteins. Constitutive activity is observed at low and high GPCR expression levels, in native systems and in recombinant systems, and has been reported for GPCRs coupled to G(s)-, G(i)- and G(q)-proteins. Constitutive activity of neurotransmitter GPCRs may provide a tonic support for basal neuronal activity. For the majority of GPCRs known to be constitutively active, inverse agonists have already been identified. Inverse agonists may be useful in the treatment of neuropsychiatric and cardiovascular diseases and of diseases caused by constitutively active GPCR mutants.

Inverse agonism at G protein-coupled receptors: (patho)physiological relevance and implications for drug discovery.

The simultaneous activation of many distinct G protein-coupled receptors (GPCRs) and heterotrimeric G proteins play a major role in various pathological conditions. Pan-inhibition of GPCR signaling by small molecules thus represents a novel strategy to treat various diseases. To better understand such therapeutic approach, we have characterized the biomolecular target of BIM-46187, a small molecule pan-inhibitor of GPCR signaling. Combining bioluminescence and fluorescence resonance energy transfer techniques in living cells as well as in reconstituted receptor-G protein complexes, we observed that, by direct binding to the Galpha subunit, BIM-46187 prevents the conformational changes of the receptor-G protein complex associated with GPCR activation. Such a binding prevents the proper interaction of receptors with the G protein heterotrimer and inhibits the agonist-promoted GDP/GTP exchange. These observations bring further evidence that inhibiting G protein activation through direct binding to the Galpha subunit is feasible and should constitute a new strategy for therapeutic intervention.

Constitutive activation of G protein-coupled receptors and diseases: Insights into mechanisms of activation and therapeutics

Structure and function of G protein-coupled receptors using NMR spectroscopy

Co-expression of guanine nucleotide-binding regulatory (G) protein-coupled receptors (GPCRs), such as the G(i/o)-coupled human 5-hydroxytryptamine receptor 1B (5-HT(1B)R), with the G(q/11)-coupled human histamine 1 receptor (H1R) results in an overall increase in agonist-independent signaling, which can be augmented by 5-HT(1B)R agonists and inhibited by a selective inverse 5-HT(1B)R agonist. Interestingly, inverse H1R agonists inhibit constitutively H1R-mediated as well as 5-HT(1B)R agonist-induced signaling in cells co-expressing both receptors. This phenomenon is not solely characteristic of 5-HT(1B)R; it is also evident with muscarinic M2 and adenosine A1 receptors and is mimicked by mastoparan-7, an activator of G(i/o) proteins, or by over-expression of Gbetagamma subunits. Likewise, expression of the G(q/11)-coupled human cytomegalovirus (HCMV)-encoded chemokine receptor US28 unmasks a functional coupling of G(i/o)-coupled CCR1 receptors that is mediated via the constitutive activity of receptor US28. Consequently, constitutively active G(q/11)-coupled receptors, such as the H1R and HCMV-encoded chemokine receptor US28, constitute a regulatory switch for signal transduction by G(i/o)-coupled receptors, which may have profound implications in understanding the role of both constitutive GPCR activity and GPCR cross-talk in physiology as well as in the observed pathophysiology upon HCMV infection.

A hallmark of G-protein-coupled receptors (GPCRs) is the conversion of external stimuli into specific cellular responses. In this tightly-regulated process, extracellular ligand binding by GPCRs promotes specific conformational changes within the seven transmembrane helices, leading to the coupling and activation of intracellular "transducer" proteins, such as heterotrimeric G proteins. Much of our understanding of the molecular mechanisms that govern GPCR activation is derived from experiments with purified receptors reconstituted in detergent micelles. To elucidate the influence of the phospholipid bilayer on GPCR activation, here we interrogated the functional, pharmacological, and biophysical properties of a GPCR, the β2-adrenergic receptor (β2AR), in high-density lipoprotein (HDL) particles. Compared with detergent-reconstituted β2AR, the β2AR in HDL particles had greatly enhanced levels of basal (constitutive) activity and displayed increased sensitivity to agonist activation, as assessed by activation of heterotrimeric G protein and allosteric coupling between the ligand-binding and transducer-binding pockets. Using 19F NMR spectroscopy, we directly linked these functional differences in detergent- and HDL-reconstituted β2AR to a change in the equilibrium between inactive and active receptor states. The contrast between the low levels of β2AR constitutive activity in cells and the high constitutive activity observed in an isolated phospholipid bilayer indicates that β2AR basal activity depends on the reconstitution system and further suggests that various cellular mechanisms suppress β2AR basal activity physiologically. Our findings provide critical additional insights into GPCR activation and reveal how dramatically reconstitution systems can impact membrane protein function.

Of 12 naturally occurring, activating mutations in the seven-transmembrane (7TM) domain of the human Ca2+ receptor (CaR) identified previously in subjects with autosomal dominant hypocalcemia (ADH), five appear at the junction of TM helices 6 and 7 between residue Ile819 and Glu837. After identifying a sixth activating mutation in this region, V836L, in an ADH patient, we studied the remaining residues in this region to determine whether they are potential sites for activating mutations. Alanine-scanning mutagenesis revealed five additional residues in this region that when substituted by alanine led to CaR activation. We also found that, whereas E837A did not activate the receptor, E837D and E837K mutations did. Thus, region Ile819-Glu837 of the 7TM domain represents a "hot spot" for naturally occurring, activating mutations of the receptor, and most of the residues in this region apparently maintain the 7TM domain in its inactive configuration. Unique among the residues in this region, Pro823, which is highly conserved in family 3 of the G protein-coupled receptors, when mutated to either alanine or glycine, despite good expression severely impaired CaR activation by Ca2+. Both the P823A mutation and NPS 2143, a negative allosteric modulator that acts on the 7TM through a critical interaction with Glu837, blocked activation of the CaR by various ADH mutations. These results suggest that the 7TM domain region Ile819-Glu837 plays a key role in CaR activation by Ca2+. The implications of our finding that NPS 2143 corrects the molecular defect of ADH mutations for treatment of this disease are also discussed.

Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract TMEM87s are eukaryotic transmembrane proteins with two members (TMEM87A and TMEM87B) in humans. TMEM87s have proposed roles in protein transport to and from the Golgi, as mechanosensitive ion channels, and in developmental signaling. TMEM87 disruption has been implicated in cancers and developmental disorders. To better understand TMEM87 structure and function, we determined a cryo-EM structure of human TMEM87A in lipid nanodiscs. TMEM87A consists of a Golgi-dynamics (GOLD) domain atop a membrane-spanning seven-transmembrane helix domain with a large cavity open to solution and the membrane outer leaflet. Structural and functional analyses suggest TMEM87A may not function as an ion channel or G-protein coupled receptor. We find TMEM87A shares its characteristic domain arrangement with seven other proteins in humans; three that had been identified as evolutionary related (TMEM87B, GPR107, and GPR108) and four previously unrecognized homologs (GPR180, TMEM145, TMEM181, and WLS). Among these structurally related GOLD domain seven-transmembrane helix (GOST) proteins, WLS is best characterized as a membrane trafficking and secretion chaperone for lipidated Wnt signaling proteins. We find key structural determinants for WLS function are conserved in TMEM87A. We propose TMEM87A and structurally homologous GOST proteins could serve a common role in trafficking membrane-associated cargo. Editor's evaluation Your work addresses the mechanisms of action of the transmembrane proteins TMEM87A and TMEM87B, which are thought to play a role in protein transport but have been implicated in other processes as well, such as signaling and acting as mechanosensitive ion channels. The study represents an important advance in the understanding of this poorly characterized family of proteins. https://doi.org/10.7554/eLife.81704.sa0 Decision letter Reviews on Sciety eLife's review process Introduction TMEM87 proteins are transmembrane proteins found in eukaryotic organisms from fungi and plants to mammals (Edgar, 2007). In humans, two paralogs have been identified: TMEM87A and TMEM87B (Edgar, 2007). TMEM87A and TMEM87B have been associated with protein transport to and from the Golgi, mechanosensitive cation channel activity, and cardiac and other developmental processes (Hirata et al., 2015; Shin et al., 2020; Russell et al., 2014; Patkunarajah et al., 2020). In humans, TMEM87 proteins have been implicated in developmental diseases and cancers (Cooper et al., 2020; Shaver et al., 2016; Yu et al., 2016; Digilio et al., 2022). Several lines of evidence support a role for TMEM87 in protein trafficking. First, TMEM87s localize to the Golgi (Hirata et al., 2015; Shin et al., 2020). Second, overexpression of TMEM87A or TMEM87B partially rescues defective endosome-to-trans-Golgi network retrograde traffic in HEK293 cells lacking the Golgi-associated retrograde protein (GARP) complex member VPS54 (Hirata et al., 2015). Third, TMEM87A has been identified as retrograde cargo captured by mitochondrial golgins (Shin et al., 2020). A recent study, however, associated TMEM87A with mechanosensitive cation channel activity (Patkunarajah et al., 2020). Deflection of micropillar culture supports resulted in cationic currents in melanoma cells that were reduced by TMEM87A expression knockdown. Additionally, TMEM87A overexpression resulted in mechanosensitive currents in PIEZO1 knockout HEK293T cells (Patkunarajah et al., 2020). TMEM87A knockout melanoma cells show increased adhesion strength and decreased migration, suggesting potential roles for TMEM87A in these processes in cancers (Patkunarajah et al., 2020). TMEM87B has been linked to developmental processes in recurrent 2q13 microdeletion syndrome (Russell et al., 2014; Yu et al., 2016; Digilio et al., 2022). Patients with this syndrome exhibit cardiac defects, craniofacial anomalies, and developmental delays. The vast majority of 2q13 microdeletions include TMEM87B (Yu et al., 2016). In zebrafish, morpholino knockdown of TMEM87B resulted in cardiac hypoplasia (Russell et al., 2014). In a patient with a severe cardiac phenotype, whole-exome sequencing uncovered a paternally inherited TMEM87B missense mutation and chromosome 2 deletion inherited from an unaffected mother (Yu et al., 2016). Both TMEM87A and TMEM87B have additionally been implicated in cancers such as non-small cell lung cancer through fusion or suspected interaction with oncogenes (Cooper et al., 2020; Shaver et al., 2016; Li et al., 2008). Bioinformatic analysis has grouped TMEM87A and TMEM87B in a small family of proteins (termed lung 7TM receptors or LUSTRs) with so-called orphan GPCRs GPR107 and GPR108 (Edgar, 2007). Like TMEM87s, GPR107, and GPR108 localize to the Golgi and have suggested roles in protein trafficking. GPR107 is implicated in the transport of Pseudomonas aeruginosa Exotoxin A, Campylobacter jejuni cytolethal distending toxin (CDT), and ricin, while GPR107 knockout cells display deficits in receptor-mediated endocytosis and recycling (Tafesse et al., 2014; Carette et al., 2011; Elling et al., 2011; Zhou et al., 2014). GPR108 is critical for the transduction of a majority of AAV serotypes (Meisen et al., 2020; Dudek et al., 2020). While a GPR107 knockout mouse was embryonic lethal, a GPR108 knockout mouse was viable and revealed a potential role for GPR108 in the regulation of Toll-like receptor-mediated signaling (Zhou et al., 2014; Dong et al., 2018). Despite being implicated in important cell biological processes and human health, our molecular understanding of TMEM87s and LUSTR proteins is limited and no experimental structures of these proteins have been reported to date. Here, we present a cryo-electron microscopy (cryo-EM) structure of human TMEM87A. Through structural and bioinformatic analyses, we find TMEM87A belongs to a broader protein family we term GOLD-domain seven transmembrane (GOST) proteins. We speculate TMEM87A and other GOST proteins could function as trafficking chaperones for membrane-associated cargo. Results Full-length human TMEM87A was expressed and purified from Sf9 insect cells and reconstituted into lipid nanodiscs composed of MSP1D1 and a mixture of DOPE, POPS, and POPC lipids (Figure 1—figure supplement 1). Cryo-EM was used to determine the structure of TMEM87A to a nominal resolution of 4.7 Å, with better resolved regions reaching ~4.3 Å in the core of the protein. The map was of sufficient quality to place secondary structure elements unambiguously and, using an AlphaFold2 predicted structure as a starting model, to place and refine 395/555 residues (Figure 1, Figure 1—figure supplements 2 and 3, Supplementary file 1). The modeled portion of TMEM87A and corresponding predicted structure are similar (overall r.m.s.d.=1.8 Å) with a minor difference in the relative position of extracellular and transmembrane regions. About 37 N-terminal residues (amino acids 1–37), 88 C-terminal residues (475-555), and 36 residues in loops within the extracellular domain (145–173, 192–198) were not resolved in the cryo-EM map and are unmodeled. Figure 1 with 3 supplements see all Download asset Open asset Structure of TMEM87A in lipid nanodiscs. (a) 4.7 Å cryo-EM map and (b) model for TMEM87A viewed from the plane of the membrane and the extracellular or lumenal side (right). The GOLD domain is colored yellow, seven-transmembrane domain colored blue, and modeled phospholipid colored pink. (c) TMEM87A with rainbow coloring from N-terminus (blue) to C-terminus (red). (d) Corresponding cartoon of domain topology with rainbow coloring from N-terminus (blue) to C-terminus (red). Positions of disulfide bonds and residues noted in the text are indicated. EM, electron microscopy. TMEM87A is composed of an extracellular beta-sandwich domain atop a G-protein coupled receptor (GPCR)-like seven-transmembrane (7TM) domain (Figure 1). The extracellular beta-sandwich domain is formed by seven beta strands in two opposing sheets, with three strands on the N-terminal face and four on the opposing face. The N-terminal face consists of β1, β3, β7, and a region between β5 and β6 lacking secondary structure that packs against β3 at the top of the sheet. The opposing face of the β-sandwich is formed from β2, β4, β5, and β6 with a helix-turn-helix motif between β4 and β5. A short 10 residue loop extends from the end of β7 to the 7TM domain at the extracellular side of H1. Two disulfide bonds are observed. The first, between C74 and C128, connects two loops at the top of this extracellular domain. The second, between C89 and C431, tethers the bottom of the extracellular domain to the top of the 7TM domain in the H6–H7 linker, perhaps constraining their relative movement. The transmembrane region of TMEM87A is structurally similar to other 7TM proteins, with a few notable differences (Figure 2, Figure 2—figure supplement 1). Using Dali to compare the isolated TMEM87A transmembrane domain to all experimentally determined protein structures returns 7TM proteins as clear structural homologs, with the fungal class D GPCR Ste2, microbial opsins, and additional class A GPCRs among the hits (Holm, 2020; Velazhahan et al., 2021; Hayashi et al., 2020; Yamamoto et al., 2009). Figure 2 with 3 supplements see all Download asset Open asset The TMEM87A transmembrane domain and comparison to an A class GPCR. (a) View of the TMEM87A model from the ‘back’ in the plane of the membrane and zoomed view highlighting the position of helix 8 with residue N465 indicated (corresponding to TMEM87B N456D implicated 2q13 deletion syndrome). (b) Overlay of TMEM87A (blue) and β1 adrenergic receptor (mgβ1AR PDB: 2Y00, orange) transmembrane domains. (c) Overlay of mgβ1AR alone (PDB: 2Y00, orange), in complex with arrestin (PDB: 6TKO, red) in complex with heterotrimeric Gi-proteins (PDB: 7S0F, pink). (d, e) Overlay of TMEM87A (blue) and (d) arrestin (red surface) or (e) heterotrimeric Gi (pink surface) from mgβ1AR complex structures(PDB: 6TKO, 7S0F). Helix 8s (H8s) are colored shades of green and denoted with asterisks. Since TMEM87A shares structural features with ion-conducting opsins and previous work implicated TMEM87A in mechanosensitive cation conduction (Patkunarajah et al., 2020), we asked whether TMEM87A displayed channel activity in isolation. We purified and reconstituted TMEM87A into proteoliposomes and recorded currents across patched membranes in response to membrane stretch induced by pressure steps. We observed neither basal nor mechanically activated currents in TMEM87A reconstituted proteoliposomes, in contrast to the mechanosensitive ion channel TRAAK used as a positive control (Figure 2—figure supplement 2). This result is consistent with the lack of a clear ion conducting path in the TMEM87A structure. We conclude that under these conditions, TMEM87A does not form a mechanosensitive ion channel. Since TMEM87A contains a 7TM domain, we next asked whether it has structural features consistent with G-protein coupled receptors. TMEM87A and Class A GPCRs have a similar transmembrane helix organization, for example, TMEM87A and the β1-adrenergic receptor are superimposed with an overall r.m.s.d. of 4.13 Å (Figure 2). Intriguingly, the position of cytoplasmic helix 8 in TMEM87A differs substantially from experimental structures of GPCRs. In TMEM87A, helix 8 turns back towards the center of the protein and packs against the bottom of the transmembrane domain (Figure 2a and b). In experimental GPCR structures, helix 8 is instead rotated nearly 180° and adopts a different position projecting toward the surrounding membrane (Warne et al., 2011; Lee et al., 2020; Alegre et al., 2021; Figure 2b–c). This position of helix 8 in GPCRs appears necessary to accommodate G-protein or arrestin binding and is similarly positioned in apo- and complexed-receptor structures (Figure 2c). The position of helix 8 in TMEM87A would sterically clash with putative G-protein or arrestin binding through interfaces analogous to those observed in GPCR structures (Figure 2c–e). Both helix 8 and the cytoplasmic surface it interacts with are highly conserved in TMEM87A across species (Figure 2—figure supplement 3). Notably, the TMEM87B point mutation implicated in 2q13 deletion syndrome (TMEM87B N456D) corresponds to TMEM87A residue N465 at the TM7-helix 8 junction (Yu et al., 2016; Figure 2a), suggesting helix 8 is important for TMEM87B function. In addition to this difference in helix 8 position, TMEM87A does not share canonical motifs of class A or D GPCRs including the class A NPxxY activation motif, PIF motif, and D(E)/RY motif or the class D LPLSSMWA activation motif (Velazhahan et al., 2021; Palczewski et al., 2000; Wang et al., 2020). Taken together, these differences suggest that TMEM87A may not couple to G proteins or arrestins like canonical GPCRs. We found the extracellular/lumenal region of TMEM87A adopts a Golgi dynamics (GOLD) domain fold (Figure 3, Figure 3—figure supplement 1). Using Dali to compare the isolated TMEM87A extracellular β-sandwich domain (residues 38–218) to all experimentally determined protein structures returns human p24 GOLD domain proteins as top hits. Superposition of the TMEM87A extracellular domain and p24 shows conservation of β strand topology throughout the domain, with differences predominantly in regions connecting β strands including the helix-turn-helix motif in the TMEM87A GOLD domain (Holm, 2020; Nagae et al., 2016; Figure 3—figure supplement 1a–c). GOLD domain proteins have established roles in the secretory pathway (Nagae et al., 2016; Anantharaman and Aravind, 2002) and p24 proteins are implicated as cargo receptors for protein transport, with the GOLD domain mediating cargo recognition (Pastor-Cantizano et al., 2016; Mendes and Costa-Filho, 2022). Among different p24 proteins, the GOLD domain loops are among the most variable regions and have been hypothesized to recognize different cargo (Nagae et al., 2016). Identification of a GOLD domain in TMEM87A suggests a structural explanation for the proposed roles of TMEM87s in protein transport, with major differences in GOLD domain loops between TMEM87A and p24 perhaps indicative of differences in interacting partners. Figure 3 with 2 supplements see all Download asset Open asset Predicted structural homology of GOST (GOLD domain seven transmembrane helix) family proteins. Experimentally determined TMEM87A structure and AlphaFold2 predicted structures of all identified human GOST family proteins (TMEM87B, GPR107, GPR108, GPR180, TMEM145, TMEM181, and WLS) shown as surfaces in the same orientation from the membrane plane. GOLD domains are yellow, seven-transmembrane domains are blue, N-terminal helices (if present) are gray, and membrane-facing slots are outlined in pink. Low confidence (<80 pLDDT) regions of predicted structures are removed for clarity (full structures are displayed in Figure 3—figure supplement 2). We next asked whether further structural comparison could provide insight into TMEM87A functions. We performed a Dali search against the entire AlphaFold2-predicted human proteome using the experimental TMEM87A structure as the reference (Holm, 2020; Jumper et al., 2021; Varadi et al., 2022). This search uncovered proteins predicted to have the same domain topology consisting of a GOLD domain fused to a 7TM domain: all four members of the LUSTR family and four previously unrecognized homologs GPR180, TMEM145, TMEM181, and Wntless (WLS) (Figure 3, Figure 3—figure supplement 1d–j, Figure 3—figure supplement 2a–g). The predicted structures are all structurally homologous to TMEM87A (overall r.m.s.d range from 1.85 to 3.87 Å). We propose an expansion of the LUSTR family to include these additional members and to name them GOST proteins (for GOLD domain seven-transmembrane helix proteins). While three newly identified GOST proteins have been minimally characterized to date (prior work has associated GPR180 with TGF-B signaling and TMEM181 with Escherichia coli CDT toxicity Balazova et al., 2021; Carette et al., 2009), WLS (also known as GPR177 or evenness interrupted homolog [EVI]) has been extensively studied. WLS plays an essential role in chaperoning lipidated and secreted Wnt signaling proteins between internal membranes and the cell surface (Bänziger et al., 2006; Bartscherer et al., 2006; Fu et al., 2009). Recent cryo-EM structures of WLS bound to Wnt3a or Wnt8a provide insight into the basis for chaperone-client interactions (Nygaard et al., 2021; Zhong et al., 2021; Figure 4a). The extracellular region of Wnt interacts extensively with the WLS GOLD domain and an extended palmitoleated hairpin of Wnt that is buried in the large hydrophobic cavity within the WLS 7TM domain. Comparison of TMEM87A with WLS shows that the transmembrane regions are well aligned with two notable differences. First, WLS H4 and H5 are shifted approximately 13 Å away from the center of the protein, resulting in a larger cavity compared to TMEM87A necessary to accommodate the insertion of the lipidated Wnt hairpin (Figure 4b). The relative orientation of the GOLD and 7TM domains in TMEM87A and WLS also differs, with the WLS GOLD domain shifted approximately 26 Å in the same direction of movement as the H4/H5 shift (Figure 4b). Intriguingly, TMEM87A and WLS exhibit similar overall patterns of surface electrostatic charge including in the 7TM cavity that, in WLS, mediates binding of the Wnt lipidated hairpin (Figure 4c–d). Figure 4 Download asset Open asset Structural comparison of TMEM87A and WLS bound to Wnt. (a) WLS-Wnt8a complex structure (PDB: 7KC4) with WLS GOLD domain light yellow, the WLS seven-transmembrane domain light blue, the N terminal helix light gray, and Wnt8a light green. (b) Overlay of TMEM87A and WLS (PDB 7KC4) experimental structures, with TMEM87A GOLD domain colored yellow and TMEM87A transmembrane domain colored blue. Zoomed in views highlight the differences in relative domain orientation between TMEM87A and WLS. (c, d) Views of (c) TMEM87A and (d) WLS electrostatic surfaces. Scale is from –10 kbTec−1 to 10 kbTec−1. Perhaps the most striking structural feature of TMEM87A is the large cavity within the 7TM domain, a feature that is similar in several respects to the cavity in WLS structures (Figure 5a and d). In TMEM87A, this cavity measures approximately 8,637 Å (Shin et al., 2020) as compared to 12,350 Å (Shin et al., 2020) for the WLS cavity. It is funnel-shaped, presenting a large opening to the extracellular side near the GOLD domain and tapering closed near the intracellular surface above helix 8. The corresponding cavity in experimental WLS structures is filled by the lipidated Wnt3a/8 a hairpin (Figure 5d). Interestingly, the predicted WLS structure shows a smaller cavity relative to experimental WLS-Wnt structures. This suggests the apo-WLS cavity could dilate to accommodate Wnt binding (Figure 3—figure supplement 2h–j). The TMEM87A cavity is also exposed to the lipid membrane. H5 and H6 are splayed apart to open a large ‘slot’ between the cavity and the upper leaflet of the bilayer (Figure 5b). Density within the membrane exposed portion of the hydrophobic cavity is consistent with a bound phospholipid. A partial lipid is modeled based on density, chemical environment, and relationship to WLS, but we note this assignment is speculative given the relatively low resolution of the reconstruction. Notably, the WLS cavity is open to the upper leaflet through a similar slot between H5 and H6 and a phospholipid is bound in a similar position in experimental structures (Figure 5d–f). The slot in WLS is presumably important because it permits access of the lipidated Wnt hairpin from the membrane into the cavity binding site. Mapping sequence conservation onto TMEM87A and WLS structures shows a similar pattern with strongly conserved residues in the transmembrane region lining the internal cavity (Figure 2—figure supplement 3). Taken together, the structural homology between TMEM87A and WLS suggests they could similarly have evolved to bind and facilitate membrane trafficking of hydrophobic cargo. Figure 5 Download asset Open asset TMEM87A and WLS cavities are exposed to solution and membrane. (a) TMEM87A structure with CASTp calculated internal cavity shown as a transparent orange surface. (b) Surface of TMEM87A with membrane-facing slot (red outline) connecting the cavity and upper leaflet of the bilayer highlighted. (c) Modeled phospholipid (pink) is shown as a surface (left) or within cryo-EM density (right). (d) WLS (PDB: 7KC4) structure with CASTp calculated internal cavity shown as a transparent yellow surface, with bound Wnt8a (pale green surface) and phospholipid (pink) shown filling the cavity. (e) Zoomed in view shows surface representation of WLS (PDB: 7KC4) with open membrane-facing slot (red outline) and phospholipid (pink). (f) Overlay of the experimental TMEM87A and WLS structures showing relative positions of the H6 membrane-facing slot and modeled phospholipids. EM, electron microscopy. Discussion In this study, we report the first experimental structure of TMEM87A and consider its potential functional roles. TMEM87A shows general structural homology to ion transporting or conducting opsins and a recent report proposed TMEM87A contributes to mechanosensitive ion channel activity in cultured cells (Patkunarajah et al., 2020). We found no evidence of channel activity from purified TMEM87A reconstituted into proteoliposomes. This observation is consistent with the structure because while TMEM87A contains a solvent exposed cavity in its 7TM domain, it is sealed to the cytoplasmic side and open to the lipid bilayer, unlike typical ion conduction pathways. We cannot exclude the possibility that TMEM87A contributes to mechanosensitive ion channel currents under other conditions, in complex with other proteins, or in a cellular context. Still, further structural analyses and relationship to WLS suggest it may rather act indirectly to impact ion channel function, perhaps through regulating transport, expression, or activity of a mechanosensitive ion channel. We discovered that TMEM87 consists of an extracellular or lumenal GOLD domain atop a membrane-spanning seven-transmembrane helix domain and shares this characteristic organization with at least seven other GOST proteins in humans. Similarity to canonical GPCRs in the transmembrane region raises the question of whether TMEM87A or other GOST proteins are competent for GPCR signaling. While ligands have been proposed for some GOST proteins, for GPR107 and for GPR180, no molecular evidence of G-protein signaling has been reported for family member and GPR180 has been reported not to as a GPCR et al., 2021; et al., et al., The position of helix 8 in TMEM87A is sterically with G-protein or arrestin binding and predicted structures of other GOST proteins present similar Taken together, this raises the possibility that GOST proteins are structurally with canonical GPCR signaling. GOST proteins not function as or functional roles they GOST family proteins are relatively previous work supports roles for members in different of protein transport (Hirata et al., 2015; Shin et al., 2020; et al., 2014; Carette et al., 2011; Elling et al., 2011; Zhou et al., 2014; et al., 2020; Dudek et al., 2020; Carette et al., et al., 2006; Bartscherer et al., The best is WLS, for which a large of work an essential role in Wnt signaling. secreted from the cell to their signaling but they are lipidated and within intracellular WLS Wnt transport and secretion by interacting with Wnt extracellular regions its GOLD and hydrophobic Wnt regions the 7TM TMEM87A and other GOST proteins or are predicted to key features suggesting they could serve analogous they have GOLD domains positioned to with extracellular domains or proteins, have large transmembrane and the cavities to the extracellular side and the membrane. This raises the possibility that GOST family proteins could transport lipidated or hydrophobic secreted proteins through a analogous to Wnt binding and transport by WLS. In this the two domains of GOST proteins would a of in which the GOLD domain and the internal cavity of cargo cargo for GOST proteins and other lipidated secreted proteins such as and some et al., 2011; and 2011; et al., 2018). are necessary to the that the GOST family proteins have common roles in transport of lipidated or proteins. Materials and methods expression, and a The sequence for TMEM87A was for expression in cells and The sequence was into a based on the with an C-terminal and a for expression of cells were used to a to the Sf9 cells were cultured in these cells were not further or for and was from cells with to the was by cells at 2 cells with at a of of with by and at was in a similar to the The was used to Sf9 cells at 2 cells at a of of cells expressed protein were by at for 10 and at A cell from 1 culture was and by in 1 and 1 1 A, 10 1 1 and 1 The membrane was by at for and with a in 1 and was with for 2 at The mixture was at for and the was bound to 5 coupled to for 2 at The was in a and with 1 1 and 2 1 and and The was in 5 of 1 with and in the TMEM87A was with an additional 8 1, to with and onto a on an in TMEM87A were and into proteoliposomes or nanodiscs. a purified TMEM87A was reconstituted into MSP1D1 nanodiscs with a mixture of lipids at a at a of et al., 2009). First, lipids in and 1 was with additional and TMEM87A. This solution was at for addition of purified The solution with MSP1D1 was at for addition of of This was at for addition of of This mixture was at 4 was of by large and with a was for 10 at onto a in corresponding to TMEM87A in MSP1D1 were and used for

Oncoprotein Networks

Cellular function, proliferation, differentiation and death are regulated through a number of extracellular stimuli which mediate the transcription of specific genes, either directly or indirectly via specific intracellular pathways (Alberts et al, 1994). In leukaemia the mediators at work are primarily haematopoietic growth factors and cytokines (Table I). These small polypeptides or glycoproteins play pivotal roles in normal and neoplastic cell proliferation by binding to their cell surface receptors and thus activating signal transduction pathways leading to gene transcription (Carpenter et al, 1999; Shuai, 1999). Three general families of cell-surface receptors have been characterized by their modes of action: ion channel-linked receptors, G-protein-linked receptors and enzyme-linked receptors. The first two families are involved in processes such as neurotransmitter signalling and are of little known significance in cytokine action. The enzyme-linked receptors, however, acting either directly as enzymes or in association with enzymes, activate intracellular events and play important roles in cytokine function. Five known classes of enzyme-linked receptors have been described: (1) receptor guanylyl cyclases, (2) receptor tyrosine phosphatases, (3) receptor serine/threonine kinases, (4) receptor tyrosine kinases, and (5) tyrosine kinase-associated receptors (Alberts et al, 1994). Most growth factors and cytokines bind to receptor tyrosine kinases and tyrosine kinase-associated receptors on the cell surface. Some tyrosine kinase-associated receptors have no inher- ent kinase activity but can regulate the haematopoietic system by activating a variety of non-receptor protein tyrosine kinases; these have been named the cytokine receptor superfamily (Bazan, 1990; Ihle et al, 1998). As aberrant signalling through these receptors and their downstream pathways plays a significant role in leukaemogenesis, their modulation will probably result in therapeutic benefits (Frank, 1999). Studies directed at understanding interferon (IFN) function have provided crucial information on the signalling pathways activated by IFNs and several other cytokines (Darnell et al, 1994; Ihle et al, 1998; Frank, 1999; Shuai, 1999). These pathways involve the activation of a family of non-receptor tyrosine kinases called Janus kinases (Jaks), and the recruitment by Jaks of another family of proteins called signal transducer and activator of transcription (Stat) that induce gene transcription (Decker, 1999; Shuai, 1999). As Jak-Stat pathways are utilized by a variety of growth factors, cytokines and hormones (Darnell et al, 1994; Taniguchi, 1995; Shuai, 1999), better understanding of their role in normal haematopoiesis and in leukaemogenesis will probably lead to more selective and less toxic therapies. This approach has already been proven to be effective in initial studies of the drug STI571 in chronic myelogenous leukaemia (CML) (Druker et al, 1999; Talpaz et al, 2000). The cytokine receptor superfamily is divided into four subfamilies on the basis of the characteristic structural motifs found in their extracellular binding domains (Fig 1A) (Bazan, 1990; Alberts et al, 1994; Taniguchi, 1995; Ihle et al, 1998). These extracellular domains are characterized by the presence of conserved motifs, suggesting their common evolution to utilize common signalling pathways. Many cytokine receptors consist of two or more subunits (named α, β and γ) with one subunit common to several cytokine receptors (Taniguchi, 1995). For example, in the gp130 subfamily, the receptors for interleukin 6 (IL-6), IL-11, oncostatin M (OSM), leukaemia inhibitory factor (LIF) and ciliary neurotropic factor (CNTF) all signal through a common βc-chain called gp130 (Fig 1B). The sharing of receptor subunits allows the cytokine receptors to initiate biological cytokine activities that are both specific and redundant (Taniguchi, 1995; Carpenter et al, 1999). The cytokine receptor superfamily. (A) Cytokine receptors subfamilies. Receptor components shared by different subtypes are shown in the same colour. For example, members of the type 1 receptor group are characterized by the presence of multiple fibronectin type III repeats in the extracellular domain. Type 1 receptors include the gp130 family, the IL-2 receptor, the gp140 family and the GH receptor. Type 2 receptors include interferon and IL-10 receptors. Type 3 receptors include members of tumour necrosis factor (TNF) and Fas receptors. Type 4 includes the IL-1 receptor. (B) The gp130 receptor family are compared with receptor tyrosine kinases (RTK). Note that some members of the RTK family, independently of Jaks, can mediate Stat activation. The receptors for IL-12, granulocyte colony-stimulating factor (G-CSF) and leptin have a similar extracellular binding domain and are therefore classed in the same family, although no clear association of these receptors with gp130 has been clearly demonstrated. All receptors of the gp130 family, with the exception of IL-12 receptor, activate Stat3. Dimerization of the cytoplasmic component of the cytokine receptor is the necessary next step in the initiation of cellular signalling (Carpenter et al, 1999). The dimerized units then associate with intracellular non-receptor tyrosine kinases such as members of the Src or the Jak families of kinases to further propagate the signal (Fig 1A). Different Src family members are associated with different receptors and phosphorylate distinct but overlapping sets of proteins (Stahl et al, 1993; Alberts et al, 1994). For example, Lck, Lyn and Fyn have been shown to be activated by IL-2 and to associate with the same region of the IL-2 receptor (Kobayashi et al, 1993). Src kinases can also associate with receptors having intrinsic kinase activity, thus accounting for the overlap seen between the pathways activated by the Src family of tyrosine kinase-associated receptors and by the receptor tyrosine kinases. The Jak family of kinases (Jak1, Jak2, Jak3 and Tyk2) includes relatively large proteins that are able to bind cytokine receptor β components. (Duhe & Farrer, 1998; Ihle et al, 1998; Yeh & Pellegrini, 1999; Ward et al, 2000). The highly conserved structure of the Jak family members consists of seven Jak homology (JH) domains (Taniguchi, 1995; Yeh & Pellegrini, 1999). The JH1 domain at the carboxy terminus of the protein includes a domain with tyrosine kinase activity (Fig 2A). In general, Jak proteins are divided into an amino-terminal region (N), a catalytically inactive kinase-like (KL) domain and the tyrosine kinase (TK) domain (Yeh & Pellegrini, 1999). The cytokines, growth factors and hormones that activate Jaks are listed in Table II. Jaks are able to associate non-covalently with the cytokine receptors through their cytoplasmic domain as well as with each other (Livnah et al, 1999; Remy et al, 1999). This association results in transphosphorylation and activation of the kinase activity of Jaks, and phosphorylation of other downstream targets (Fig 2B). The Jak-Stat pathway. (A) Domains of Jak protein. JH1 is the catalytic domain with tyrosine kinase (TK) activity. KL is a catalytically inactive kinase-like domain with unknown function. SOCS are suppressors of cytokine signalling which inhibit Jaks. RBR is the receptor binding region. 'SH2' is a SH2-like domain with unidentified function. (B) Mechanism of Stat activation by cytokine receptors and their negative regulation by SOCS. Ligand binding and receptor dimerization result in Jak activation, Stat dimerization, and translocation into the nucleus and transcription of genes including those coding for SOCS. The latter have a negative regulatory effect on the pathway. The crucial role of Jaks in cytokine signalling has been established in mouse knockout models. Newborn mice with homozygously disrupted Jak1 are small, die early, and are profoundly deficient in thymocyte production and B-lymphocyte differentiation (Rodig et al, 1998). Jak2-deficient mice embryos do not respond to various haematopoietic cytokines, respond poorly to colony-stimulating factor 1 (CSF-1) and stem cell factor (SCF), and die of severe anaemia as a result of ineffective erythropoiesis (Neubauer et al, 1998; Parganas et al, 1998). Jak3-deficient mice have profoundly defective immune systems characterized by a hypoplastic thymus, non-functional peripheral T cells and a lack of natural killer cells among other defects (Thomis et al, 1995; Eynon et al, 1996). Several disorders are associated with Jak-related abnormalities. These include severe combined immunodeficiency (SCID) due to Jak3 mutations (Notarangelo, 1996), leukaemia due to chromosomal translocations resulting in TEL/JAK2 constructs causing constitutive Stat5 activation, and IL-3-independent cellular proliferation (Lacronique et al, 1997; Peeters et al, 1997). Furthermore, infection with human T-lymphotropic virus 1 (HTLV-1) and Abelson murine leukaemia viruses results in enhanced tyrosine kinase activity of Jaks, which may account for their leukaemogenic potential (Danial et al, 1995; Migone et al, 1995). Jak proteins interact with a number of intracellular signalling proteins, most of which undergo tyrosine phosphorylation upon cytokine stimulation and are therefore likely to be substrates and downstream effectors of Jaks (Table III). Although the biological effects of most of these interactions are still unknown, it is hypothesized that Jaks regulate many cytokine signalling events. The Stat proteins were first identified in the late 1980s in studies aimed at understanding the molecular mechanisms of IFN-mediated effects. These studies identified a number of genes whose transcription was induced by IFN-α and IFN-γ. Specific DNA elements were found to bind proteins such as interferon-α-stimulated gene factor-3 (ISGF-3) (Fu et al, 1992; Khan et al, 1993). These factors, located in the cytoplasm, are rapidly translocated to the nucleus and bind to their cognate DNA sites upon IFN stimulation (Shuai et al, 1992). ISGF-3 consists of three proteins: p48, p91/p84 and p113. The latter two are related proteins also known as Stat1α/β and Stat 2 respectively (Schindler et al, 1992). Five additional Stat proteins (Stat3, Stat4, Stat5A, Stat5B and Stat6) have been identified and their activation by various cytokines documented (Table II) (Hou et al, 1994; Yamamoto et al, 1994; Zhong et al, 1994; Mui et al, 1995). Stats are transcription factors whose activities are modified and regulated by tyrosine phosphorylation (Shuai et al, 1993a; Darnell, 1997). This process has been shown to be necessary for Stat homo- or heterodimerization, translocation to the nucleus, DNA binding and gene activation (Fig 2B) (Shuai et al, 1994; Zhong et al, 1994). All Stat proteins share conserved domains important for their function (Shuai, 1999). The SH2 domain is necessary for Stat tyrosine phosphorylation (Shuai et al, 1993b). The binding of a cytokine to the α subunit of the cytokine receptor rapidly induces tyrosine phosphorylation of the cytoplasmic domains of the receptor β subunit by activated Jak kinases, thus providing a docking site for Stat proteins (as well as other SH2-containing proteins such as the growth factor receptor binding protein 2, or Grb2). The SH2 domain of a Stat protein is thought to bind the tyrosine-phosphorylated residue of the cytokine β receptor with specific cytokine receptors activating specific Stats (Uddin et al, 1995; Yan et al, 1996). The SH2 domains are also necessary for Stat dimer formation (Heim et al, 1995; Carpenter et al, 1999; Decker, 1999; Shuai, 1999) and the N-terminal region of the Stat molecule for DNA binding (Vinkemeier et al, 1996; Xu et al, 1996). The differential DNA binding affinities of different Stat proteins are important in achieving specificity in Stat-mediated gene activation. To induce gene transcription, Stat proteins interact with other transcription factors such as the p300/CREB binding protein (CBP) family of co-activators, which requires histone acetyltransferase activity (Bhattacharya et al, 1996; Horvai et al, 1997; Decker & Kovarik, 1999; Heim, 1999). The transcriptional activity of Stats can also bemodulated by phosphorylation of their serine and threonine residues, although the mechanistic implications of such regulation are not entirely clear (Eilers et al, 1995; Decker, 1999). While serine phosphorylation is not sufficient to induce Stat activation, it can enhance it. Kinases involved in this process include Jun NH-2 kinase (JNK), mitogen-activated protein kinase (MAPK), protein kinase C and protein kinase A (Frank, 1999). The function of various Stats has been elucidated through the creation of mutant mice with disrupted Stat genes (Levy, 1999; Ward et al, 2000). Mice deficient in Stat1 are extremely sensitive to viral infections, consistent with Stat1's presumed importance in IFN function. (Durbin et al, 1996; Meraz et al, 1996) Mice deficient in Stat3, however, demonstrate early embryonic lethality (Takeda et al, 1997). Stat4-deficient mice have no developmental abnormalities but are completely resistant to IL-12 stimulation and fail to induce Th1 polarization of T-helper lymphocytes (Kaplan et al, 1996a; Thierfelder et al, 1996). Stat5A/B knockout mice develop normally except for defects in adult mammary gland development and lactogenesis (Liu et al, 1997; Udy et al, 1997). Stat6-deficient mice are not overtly abnormal but do exhibit defects in IL-4 function (e.g. a complete lack of IL-4-induced Th2 polarization of T-helper cells) (Shimoda et al, 1996). Stat activation and signalling are involved in diverse and sometimes opposite cellular events affecting growth, differentiation and apoptosis (Schindler, 1998; Frank, 1999; Mui, 1999). Stat activation, for example, may result in both growth arrest and cellular proliferation. Stat1 mediates the growth-inhibitory effects of IFN-γ by inducing the cyclin-dependent kinase inhibitor p21waf1, whereas Stat5 induces the proliferative effects of IL-3 and granulocyte–macrophage colony-stimulating factor (GM-CSF) (Chin et al, 1996; Mui et al, 1996). Similarly, in myeloid cells, phosphorylation of Stat3 can lead to IL-6 and IL-10-induced growth arrest (via induction of the cyclin-dependent kinase inhibitor p19INK4d) on the one hand and to GM-CSF- and IL-3-induced proliferation on the other (Mui et al, 1996; Chaturvedi et al, 1998; O'Farrell et al, 1998). Whether these diverse effects are tissue-specific or mediated by other factors is yet to be determined. Stats also mediate transcriptional events associated with cellular differentiation (Mui, 1999). Stat3 and Stat5 have been implicated in myeloid differentiation and Stat4 and Stat6 in Th cell differentiation. Stat3 is involved in IL-6-induced differentiation of M1 myeloid leukaemia cells, but suppresses differentiation in other cell lines (Yamanaka et al, 1996). Stat5 has been implicated in cell differentiation in a variety of systems, including thrombopoietin-dependent maturation of megakaryocytes and erythropoietin-dependent maturation of erythroid cell lines (Mui, 1999). When activated by IL-4, Stat6 helps regulate the expression of cell-surface proteins such as major histocompatibility complex (MHC) class II antigens, immunoglobulin class switching and Th2 differentiation (Rothman et al, 1991; Kaplan et al, 1996b). Therefore, Stat activation may play a key role in overcoming differentiation arrest. Stats are also important in regulating programmed cell death. In fact, constitutive expression of caspases necessary for induction of apoptosis in fibroblasts may depend on Stat1 (Kumar et al, 1997). For example, reconstitution of Stat1 in Stat1-null U3A cells, which do not respond to tumour necrosis factor α (TNF-α), restores basal caspase expression and sensitivity to TNF-induced apoptosis (Kumar et al, 1997). Conversely, Stat3 and Stat5 contribute to the antiapoptotic effects of IL-6 and IL-2 respectively (Fukada et al, 1996; Zamorano & Keegan, 1998). In response to IFN-γ, Stat1 mediates the upregulated expression of Fas expression and FasL, thereby activating the caspase cascade (Xu et al, 1998). Activation of the Jak-Stat pathway is both rapid and transient. Following stimulation by IFNs, Stat1 phosphorylation peaks within 15–30 min and returns to basal levels after 1–2 h. Three possible mechanisms of Stat inactivation following stimulation have been postulated: proteasome-mediated degradation, tyrosine dephosphorylation, and inhibition by various inhibitory proteins (Shuai, 1999). The first mechanism involves the ubiquitin-proteasome pathway, which is responsible for the degradation of many proteins involved in fundamental cellular processes (Kim & Maniatis, 1996). The second mechanism, tyrosine dephosphorylation, involves cytoplasmic phosphatases such as SH-2-domain-containing protein tyrosine phosphatase-1 (SHP)-1 that are believed to inhibit cytokine-activated Jak-Stat pathways through their dephosphorylation and inactivation of Jak (Hilton, 1999; Starr & Hilton, 1999). SHP-1-deficient mice demonstrate multiple haematopoietic abnormalities, including hyperproliferation and abnormal activation of granulocytes and macrophages in the lungs and in the skin, resulting in the characteristic moth-eaten appearance of their coats (Jiao et al, 1997; Takahashi et al, 1998). They also produce fewer B-cell progenitors and natural killer cells and exhibit premature thymic involution leading to immune deficiency (Jiao et al, 1997). This suggests that SHP-1 is a negative regulator of both myeloidand lymphoid lineages (Van Zant & Shultz, 1989; Yoshimura et al, 1990; de la Chapelle et al, 1993; Klingmuller et al, 1995; Furukawa et al, 1997). Ofparticular interest is a report on families with polycythaemia in whom the C-terminal region of the erythropoietin (EPO) receptor (which includes the docking site for SHP-1) is deleted (de la Chapelle et al, 1993; Furukawa et al, 1997). The third mechanism of Stat inactivation involves various inhibitory proteins including the suppressors of cytokine signalling (SOCS) and the protein inhibitors of activated Stats (PIAS) (Alexander et al, 1999; Shuai, 1999; Starr & Hilton, 1999). The SOCS proteins (eight in number) are alternatively known as JAB (Jak-binding protein), SSI (Stat-induced Stat inhibitor) or CIS (cytokine-inducible SH2-containing protein) (Yoshimura et al, 1995; Endo et al, 1997; Naka et al, 1997; Starr et al, 1997). Recent studies have established the SOCS proteins as negative regulators of cytokines (Yoshimura et al, 1995; Endo et al, 1997; Naka et al, 1997; Adams et al, 1998; Song & Shuai, 1998; Nicholson et al, 1999). Several lines of evidence also suggest that Stats are important in the induction of SOCS expression, although it is likely that Stat proteins are capable of inducing the expression of several SOCS and that each SOCS protein is capable of being induced by several Stats (Hilton, 1999). The PIAS family includes five proteins with significant sequence homology and several highly conserved domains (Chung et al, 1997; Liu et al, 1998). They are specific inhibitors of the Stat signalling pathway, with PIAS1 and PIAS3 being associated with Stat1 and Stat3 respectively (Shuai, 1999). The IFN-induced phosphorylation of Stat1 on Tyr701 is necessary for the PIAS–Stat1 interaction. Thus, the interaction of PIAS-Stat is highly specific and dependent on cytokine stimulation (Shuai, 1999). As cellular signalling pathways are intimately involved in the physiological regulation of cell survival, proliferation and differentiation, their abnormal function can lead to neoplastic changes. Studies in the Drosophila fly, which expresses a Jak and a Stat homologue, have shown that gain-of-function mutations affecting Jak cause leukaemia-like haematopoietic defects (Harrison et al, 1995). Over recent years, several investigators have reported that inappropriate activation of Stats may have a significant role in the pathogenesis of human malignancies and leukaemia in particular (Table IV) (Frank, 1999). This inappropriate activation is initiated by the abnormal hyperfunction or mutation of physiological Stat activators (such as Jaks) or by mutation of kinases not usually involved in Stat phosphorylation (such as abl), thus causing increased cellular proliferation (Frank, 1999). Various activated cellular tyrosine kinases phosphorylate Stats, either directly or via Jak activation, and induce cellular transformation. Activation of Jak1 by the oncogenic tyrosine kinase v-abl induces malignant transformation of haematopoietic cells, whereas constitutive Stat activation by mutated members of the Src kinase family induces cellular transformation (Danial et al, 1998; Frank, 1999). For example, v-src, v-fps and v-sis activate Stat3 and induce fibroblast transformation (Yu et al, 1995; Chaturvedi et al, 1998). Similarly, Lck, another Src tyrosine kinase, is overexpressed in a mouse T-cell lymphoma and is associated with Stat3, Stat5, Jak1 and Jak2 activation (Yu et al, 1997). Stat activation plays a key role in the pathogenesis of human leukaemia. Stat5 and Stat1 for example are constitutively activated in acute lymphoblastic leukaemia (ALL) and Stat1, Stat3, and Stat5 in acute myelogenous leukaemia (AML) (Gouilleux-Gruart et al, 1996; Weber-Nordt et al, 1996; Xia et al, 1998). The chromosomal translocation t(9:12)(p24:p13) found in acute lymphoblastic leukaemia (ALL) results in the fusion of the helix-loop-helix domain of the transcription factor of the family of transcription with the catalytic domain of Jak2, thus activating Jak2 and inducing gene transcription et al, 1998; et al, 1999). of the fusion protein cells growth factor and of mice with a this protein induces a et al, 2000). Activation of these pathways is also crucial in the pathogenesis of chronic myelogenous leukaemia of gene constructs into haematopoietic cell lines induces growth proliferation & transformation of haematopoietic cells to tyrosine phosphorylation of Stat1 and Stat5 in cell lines and cells with & 1996; et al, 1996; et al, 1997). As these cells do not demonstrate Jak activation, it is likely that the enhanced kinase activity of is directly responsible for Stat phosphorylation and cellular transformation (Fig In fact, recent on the activity of the specific kinase inhibitor STI571 and to induce and in have provided evidence for the role of Stat signalling in the pathogenesis of this and suggest that inhibition of Stat activity may be (Druker et al, of on signal transduction pathways implicated in mechanisms include activation of Stat1 and Stat5, activation of pathway through and activation of via proteins such as growth factor and virus and the and protein may also the complex function through a et chromosomal translocations leading to the fusion of kinases and transcription factors include and (Frank, 1999). In the of activation of the receptor tyrosine kinase is mediated by the domain of the transcription factor leading to constitutive Stat activation and growth of haematopoietic cells et al, 1996; et al, 1997; et al, 1998). Although cellular proliferation associated with Jak-Stat pathway is seen in acute and no constitutive tyrosine phosphorylation of Stats has been seen to in chronic leukaemia et al, 1997). serine phosphorylation of Stat1 and Stat3, which is known to their function, has been seen in cells but not in normal peripheral lymphocytes et al, 1997). This serine which can enhance the transcriptional response mediated by tyrosine-phosphorylated Stats and lead to of B-cell function, may the of (Frank, 1999). viruses may also play a role in Stat activation and In the example, T cells with IL-2 and exhibit enhanced Stat3 and Stat5 activity et al, 1995). In with adult T-cell Stat1, Stat3 Stat5, as well as Jak kinases, are constitutively activated et al, 1995; Xu et al, 1995; et al, 1997). Although the mechanisms of Jak activation in cells are still unknown, it has been hypothesized that a viral protein with the IL-2 receptor to activate the pathway. the effect of on T cells is in T cells et al, 1999). Receptor tyrosine kinases are enzymes with an extracellular a and a highly conserved intracellular domain that mediates phosphorylation of tyrosine on substrates in a similar to the Jak-Stat pathway 1996; & 1998). These receptors are not activated by binding but also by such as interactions via cell in response to cellular and by stimulation of receptors et al, 1997). have been into four classes on the basis of their structural class includes the receptors for growth factor class those for growth class those for growth factor colony-stimulating factor or receptor and tyrosine receptor and class those for fibroblast growth factor (Fig et al, 1991; 1990; 1996). and significant homology in their tyrosine kinase domains but less homology in their extracellular domains 1996). All cells levels of and most et al, 1995; et al, 1995; 1996). Several studies have the role of class III receptors and their in the regulation of survival, proliferation and maturation of early haematopoietic usually in with other cytokines 1990; et al, 1991; et al, 1995; et al, 1995; et al, 1995). Several have the role of these growth factors in the pathogenesis of leukaemia and et al, 1992; et al, 1993; et al, 1995; et al, 1996; 1996; et al, 1997; et al, 1999; et al, 1999). is at levels by and lymphoid or cells, but at levels by cells 1996). levels have been shown to with in leukaemia cell growth in et al, 1999; et al, 1999). mutation of has been reported in with disorders et al, and activation of by has been shown to the proliferation of some human leukaemia cell lines et al, 1993). In expression has been reported in all subtypes of and in myeloid but not in et al, 1996; et al, 1998; et al, 1999). A large number of intracellular signalling proteins bind the on the activated including the protein and non-receptor (Alberts et al, 1994; & 1998; et al, 2000). The activation of these proteins then serine/threonine phosphorylation resulting in activation of transcription factors and modulation of cellular processes by gene transcription et al, 2000). Many major cellular signalling pathways involve tyrosine phosphorylation of Src and Jak kinases 1998). several other major such as the signalling involve activation of serine/threonine kinases et al, 2000). The proteins to the large superfamily of which also several other subfamilies including the and proteins involved in cell surface receptors to the and the family involved in the of intracellular (Alberts et al, 1994; & 1999). The three human genes and four that to the surface of the and function as et al, 1998). proteins are to a number of signalling pathways including those mediated by non-receptor tyrosine kinase-associated receptors and

Biogenic amine receptors mediate wide-ranging hormonal and modulatory functions in vertebrates, but are largely unknown in primitive invertebrates. In a representative of the most basal multicellular animals possessing a nervous system, the cnidarian Renilla koellikeri, aminergic-like receptors were previously characterized pharmacologically and found to engender control of the animal's bioluminescent and peristaltic reactions. Using degenerate oligonucleotides in a RT-PCR strategy, we obtained a full-length cDNA encoding a polypeptide with typical G protein-coupled receptor (GPCR) characteristics and which displayed a significant degree of sequence similarity (up to 45%) to biogenic amine receptors, particularly dopamine and adrenergic receptors. The new receptor, named Ren1, did not resemble any one specific type of amine GPCR and thus could not be identified on the basis of sequence. Ren1 was expressed transiently and stably in cultured mammalian cells, as demonstrated by immunocytochemistry and western blotting. Functional analysis of transfected HEK293, LTK- and COS-7 cells, based on both cAMP and Ca2+ signalling assays, revealed that Ren1 was not activated by any of the known biogenic amines tested and several related metabolites. The results indicated, however, that cells stably expressing Ren1 contained, on average, an 11-fold higher level of cAMP than the controls, in the absence of agonist stimulation. The high basal cAMP levels were shown to be specific for Ren1 and to vary proportionally with the level of Ren1 expressed in the transfected cells. Taken together, the data suggested that Ren1 was expressed as a constitutively active receptor. Its identification provides a basis for examination of the early evolutionary emergence of GPCRs and their functional properties.

G protein-coupled receptors (GPCRs) are the largest class of membrane proteins involved in signal transduction and are characterized by seven transmembrane domain architecture interconnected by extra- and intracellular loops. These loops, along with the N- and C-terminal domains, constitute the extramembranous regions in GPCRs. These regions, accounting for ~ 40% or more amino acid residues across different GPCR classes, are distinct from the conserved transmembrane domains in terms of nonconservation of sequence, diversity in length, and conformational heterogeneity. Due to technical challenges in exploring the molecular basis underlying the relation between structure, dynamics, and function in these regions, their contribution to GPCR organization and signaling remain underappreciated. Despite existingliterature on the involvement of GPCR loops in numerous aspects of GPCR biology, the functional relevance of GPCR loops in the context of their inherent conformational heterogeneity and probable membrane interaction are not well understood. This review focuses on highlighting these aspects of GPCR extramembranous regions in the overall context of GPCR organization, dynamics, and biology. We envision that a judicious combination of insights obtained from structured transmembrane domains and disordered extramembranous regions in GPCRs would be crucial in arriving at a comprehensive understanding of GPCR structure, function, and dynamics, thereby leading to efficient drug discovery.

Signalosomes: delivering cardioprotective signals from GPCRs to mitochondria

A major, unresolved question in signal transduction by G protein coupled receptors (GPCRs) is to understand how, at atomic resolution, a GPCR activates a G protein. A step toward answering this question was made with the determination of the high-resolution structure of rhodopsin; we now know the intramolecular interactions that characterize the resting conformation of a GPCR. To what degree does this structure represent a structural paradigm for other GPCRs, especially at the cytoplasmic surface where GPCR-G protein interaction occurs and where the sequence homology is low among GPCRs? To address this question, we performed NMR studies on approximately 35-residue-long peptides including the critical second intracellular loop (i2) of the alpha 2A adrenergic receptor (AR) and of rhodopsin. To stabilize the secondary structure of the peptide termini, 4-12 residues from the adjacent transmembrane helices were included and structures determined in dodecylphosphocholine micelles. We also characterized the effects on an alpha 2A AR peptide of a D130I mutation in the conserved DRY motif. Our results show that in contrast to the L-shaped loop in the i2 of rhodopsin, the i2 of the alpha 2A AR is predominantly helical, supporting the hypothesis that there is structural diversity within GPCR intracellular loops. The D130I mutation subtly modulates the helical structure. The spacing of nonpolar residues in i2 with helical periodicity is a predictor of helical versus loop structure. These data should lead to more accurate models of the intracellular surface of GPCRs and of receptor-mediated G protein activation.