Identification of residues that contribute to receptor activation through the analysis of compensatory mutations in the G protein-coupled alpha-factor receptor

The alpha-factor receptor (Ste2p) stimulates mating of the yeast Saccharomyces cerevisiae. Ste2p belongs to the large family of G protein-coupled receptors that are characterized by seven transmembrane alpha-helices. Receptor activation is thought to involve changes in the packing of the transmembrane helix bundle. To identify residues that contribute to Ste2p activation, second-site suppressor mutations were isolated that restored function to defective receptors carrying either an F204S or Y266C substitution which affect residues at the extracellular ends of transmembrane domains 5 and 6, respectively. Thirty-five different suppressor mutations were identified. On their own, these mutations caused a range of phenotypes, including hypersensitivity, constitutive activity, altered ligand binding, and loss of function. The majority of the mutations affected residues in the transmembrane segments that are predicted to face the helix bundle. Many of the suppressor mutations caused constitutive receptor activity, suggesting they improved receptor function by partially restoring the balance between the active and inactive states. Analysis of mutations in transmembrane domain 7 implicated residues Ala281 and Thr282 in receptor activation. The A281T and T282A mutants were supersensitive to S. cerevisiae alpha-factor, but were defective in responding to a variant of alpha-factor produced by another species, Saccharomyces kluyveri. The A281T mutant also displayed 8.7-fold enhanced basal signaling. Interestingly, Ala281 and Thr282 are situated in approximately the same position as Lys296 in rhodopsin, which is covalently linked to retinal. These results suggest that transmembrane domain 7 plays a role in receptor activation in a wide range of G protein-coupled receptors from yeast to humans.     

Rods are rods and cones cones, and (never) the twain shall meet.

More than 100 photopigment G protein-coupled receptors (opsins) have been sequenced and organized into six classes. Rod photoreceptors in various species have been found to express an opsin from one of the two rhodopsin classes, while cones express an opsin from one of the four remaining classes. It has now been discovered that salamander short-wavelength sensitive cones and green rods express the same opsin, while manifesting other features that classically distinguish rods from cones.     

The activation mechanism of chemokine receptor CCR5 involves common structural changes but a different network of interhelical interactions relative to rhodopsin

In G protein-coupled receptors (GPCRs), the interaction between the cytosolic ends of transmembrane helix 3 (TM3) and TM6 was shown to play an important role in the transition from inactive to active states. According to the currently prevailing model, constructed for rhodopsin and structurally related receptors, the arginine of the conserved "DRY" motif located at the cytosolic end of TM3 (R3.50) would interact with acidic residues in TM3 (D/E3.49) and TM6 (D/E6.30) at the resting state and shift out of this polar pocket upon agonist stimulation. However, 30% of GPCRs, including all chemokine receptors, contain a positively charged residue at position 6.30 which does not support an interaction with R3.50. We have investigated the role of R6.30 in this receptor family by using CCR5 as a model. R6.30D and R6.30E substitutions, which allow an ionic interaction with R3.50, resulted in an almost silent receptor devoid of constitutive activity and strongly impaired in its ability to bind chemokines but still able to internalize. R6.30A and R6.30Q substitutions, allowing weaker interactions with R3.50, preserved chemokine binding but reduced the constitutive activity and the functional response to chemokines. These results indicate that the constitutive and ligand-promoted activity of CCR5 can be modified by modulating the interaction between the DRY motif in TM3 and residues in TM6 suggesting that the overall structure and activation mechanism are well conserved in GPCRs. However, the molecular interactions locking the inactive state must be different in receptors devoid of D/E6.30.     

A 5-HT4 receptor transmembrane network implicated in the activity of inverse agonists but not agonists

Activation of G protein-coupled receptors is thought to involve disruption of intramolecular interactions that stabilize their inactive conformation. Such disruptions are induced by agonists or by constitutively active mutations. In the present study, novel potent inverse agonists are described to inhibit the constitutive activity of 5-HT(4) receptors. Using these compounds and specific receptor mutations, we investigated the mechanisms by which inverse agonists may reverse the disruption of intramolecular interactions that causes constitutive activation. Two mutations (D100(3.32)A in transmembrane domain (TMD)-III and F275(6.51)A in TMD-VI) were found to completely block inverse agonist effects without impairing their binding properties nor the molecular activation switches induced by agonists. Based on the rhodopsin model, we propose that these mutated receptors are in equilibrium between two states R and R* but are unable to reach a third "silent" state stabilized by inverse agonists. We also found another mutation in TMD-VI (W272(6.48)A) that stabilized this silent state. This mutant remained fully activated by agonists. Molecular modeling indicated that Asp-100, Phe-275, and Trp-272 might constitute a network required for stabilization of the silent state by the described inverse agonists. However, this network is not necessary for agonist activity.     

Molecular mechanisms of cation transport by the renal Na+-K+-Cl- cotransporter: structural insight into the operating characteristics of the ion transport sites

Two variants of the renal Na(+)-K(+)-Cl(-) cotransporter (NKCC2), called NKCC2A and NKCC2F, display marked differences in Na(+), Rb(+), and Cl(-) affinities, yet are identical to one another except for a 23-residue membrane-associated domain that is derived from alternatively spliced exons. The proximal portion of these exons is predicted to encode the second transmembrane domain (tm2) in the form of an alpha-helix, and the distal portion, part of the following connecting segment (cs1a). In recent studies, we have taken advantage of the A-F differences in kinetic behavior to determine which regions in tm2-cs1a are involved in ion transport. Functional characterizations of chimeras in which tm2 or cs1a were interchanged between the variants showed that both regions are important in specifying ion affinities, but did not allow delineating the contribution of individual residues. Here, we have extended these structure-function analyses by studying additional mutants in which variant residues between A and F were interchanged individually in the tm2-cs1a region (amino acid number 216, 220, 223, 229, or 233 in NKCC2). None of the substitutions were found to affect K(m (C1-)), suggesting that the affinity difference for anion transport is conveyed by a combination of variant residues in this domain. However, 2 substitutions in the tm2 of F were found to affect cation constants specifically; interestingly, one of these mutations (residue 216) only affected K(m (Rb+)) while the other (residue 220) only affected K(m (Na+)). We have thus identified two novel residues in NKCC2 that play a key role in cation transport. Because such residues should be adjacent to one another on the vertical axis of the tm2 alpha-helix, our results imply, furthermore, that the ion transport sites in NKCC2 could be physically linked.     

Constitutive activation of G protein-coupled receptors as a result of selective substitution of a conserved leucine residue in transmembrane helix III

Whereas numerous mutations of the human lutropin receptor (hLHR) and human TSH receptor (hTSHR) have been shown to cause constitutive activation of these receptors, it has been suggested that either the hFSHR as a whole, or the i3/TM VI region of the hFSHR, is less susceptible to mutation-induced constitutive activation. However, as shown herein, substitution of a highly conserved leucine residue in transmembrane III (TM III) of the hFSHR (Leu 111.18) with arginine causes a 5-fold increase in basal cAMP in transfected cells, consistent with a strong constitutive activation of the hFSHR. Interestingly, this mutant is unresponsive to further hormonal stimulation. Substitutions of hFSHR(L460) with lysine, alanine, or aspartate show that only arginine causes constitutive activation. However, all result in decreased FSH responsiveness, suggesting a role for L460 in FSH-stimulated cAMP production by the hFSHR. Because Leu 111.18 is highly conserved in rhodopsin-like G protein-coupled receptors (GPCRs), we tested the effects of substitution of the comparable leucine in the human beta2-adrenergic receptor (hbeta2-AR). Substitution of L124 in the hbeta2-AR with arginine, lysine, or alanine resulted in constitutive activation as evidenced by increased basal levels of cAMP that could be attenuated by an inverse agonist. In all cases, isoproterenol-stimulated cAMP was unaffected. Taken altogether, our data support a model whereby Leu 111.18 may play a general role in GPCRs by stabilizing them in an inactive state. Constitutive activation may arise by both a disruption of Leu 111.18 as well as the introduction of a specific residue that serves to stabilize the active state of the receptor.     

Modeling and mutagenesis of the binding site of Calhex 231, a novel negative allosteric modulator of the extracellular Ca(2+)-sensing receptor

A model of the Ca2+-sensing receptor (CaSR) seven transmembrane domains was constructed based on the crystal structure of bovine rhodopsin. This model was used for docking (1S,2S,1'R)-N1-(4-chlorobenzoyl)-N2-[1-(1-naphthyl)ethyl]-1,2-diaminocyclohexane (Calhex 231), a novel potent negative allosteric modulator that blocks (IC50 = 0.39 microm) increases in [3H]inositol phosphates elicited by activating the human wild-type CaSR transiently expressed in HEK293 cells. In this model, Glu-8377.39 plays a pivotal role in anchoring the two nitrogen atoms of Calhex 231 and locating the aromatic moieties in two adjacent hydrophobic pockets delineated by transmembrane domains 3, 5, and 6 and transmembrane domains 1, 2, 3, and 7, respectively. To demonstrate its validity, we have mutated selected residues and analyzed the biochemical and pharmacological properties of the mutant receptors transfected in HEK293 cells. Two receptor mutations, F684A3.32 and E837A7.39, caused a loss of the ability of Calhex 231 to inhibit Ca2+-induced accumulation of [3H]inositol phosphates. Three other mutations, F688A3.36, W818A6.48, and I841A7.43, produced a marked increase in the IC50 of Calhex 231 for the Ca2+ response, whereas L776A5.42 and F821A6.51 led to a decrease in the IC50. Our data validate the proposed model for the allosteric interaction of Calhex 231 with the seven transmembrane domains of the CaSR. Interestingly, the residues at the same positions have been shown to delimit the antagonist-binding cavity of many diverse G-protein-coupled receptors. This study furthermore suggests that the crystal structure of bovine rhodopsin exhibits sufficient mimicry to the ground state of a very divergent class 3 receptor to predict the interaction of antagonists with the heptahelical bundle of diverse G-protein-coupled receptors.     

Comparison of class A and D G protein-coupled receptors: common features in structure and activation

All G protein-coupled receptors (GPCRs) share a common seven TM helix architecture and the ability to activate heterotrimeric G proteins. Nevertheless, these receptors have widely divergent sequences with no significant homology. We present a detailed structure-function comparison of the very divergent Class A and D receptors to address whether there is a common activation mechanism across the GPCR superfamily. The Class A and D receptors are represented by the vertebrate visual pigment rhodopsin and the yeast alpha-factor pheromone receptor Ste2, respectively. Conserved amino acids within each specific receptor class and amino acids where mutation alters receptor function were located in the structures of rhodopsin and Ste2 to assess whether there are functionally equivalent positions or regions within these receptors. We find several general similarities that are quite striking. First, strongly polar amino acids mediate helix interactions. Their mutation generally leads to loss of function or constitutive activity. Second, small and weakly polar amino acids facilitate tight helix packing. Third, proline is essential at similar positions in transmembrane helices 6 and 7 of both receptors. Mapping the specific location of the conserved amino acids and sites of constitutively active mutations identified conserved microdomains on transmembrane helices H3, H6, and H7, suggesting that there are underlying similarities in the mechanism of the widely divergent Class A and Class D receptors.     

Pteropsin: a vertebrate-like non-visual opsin expressed in the honey bee brain

Insects have excellent color vision based on the expression of different opsins in specific sets of photoreceptive cells. Opsins are members of the rhodopsin superfamily of G-protein coupled receptors, and are transmembrane proteins found coupled to light-sensitive chromophores in animal photoreceptors. Diversification of opsins during animal evolution provided the basis for the development of wavelength-specific behavior and color vision, but with the exception of the recently discovered non-visual melanopsins, vertebrate and invertebrate opsins have generally been viewed as representing distinct lineages. We report a novel lineage of insect opsins, designated pteropsins. On the basis of sequence analysis and intron location, pteropsins are more closely related to vertebrate visual opsins than to invertebrate opsins. Of note is that the pteropsins are missing entirely from the genome of drosophilid flies. In situ hybridization studies of the honey bee, Apis mellifera, revealed that pteropsin is expressed in the brain of this species and not in either the simple or compound eyes. It was also possible, on the basis of in situ hybridization studies, to assign different long wavelength opsins to the compound eyes (AmLop1) and ocelli (AmLop2). Insect pteropsin might be orthologous to a ciliary opsin recently described from the annelid Platynereis, and therefore represents the presence of this vertebrate-like light-detecting system in insects.     

Functional analysis of the murine cytomegalovirus chemokine receptor homologue M33: ablation of constitutive signaling is associated with an attenuated phenotype in vivo

The murine cytomegalovirus (MCMV) M33 gene is conserved among all betaherpesviruses and encodes a homologue of seven-transmembrane receptors (7TMR) with the capacity for constitutive signaling. Previous studies have demonstrated that M33 is important for MCMV dissemination to or replication within the salivary glands. In this study, we probed N- and C-terminal regions of M33 as well as known 7TMR signature motifs in transmembrane (TM) II and TM III to determine the impact on cell surface expression, constitutive signaling, and in vivo phenotype. The region between amino acids R³⁴⁰ and A³⁵³ of the C terminus was found to be important for CREB- and NFAT-mediated signaling, although not essential for phosphatidylinositol turnover. Tagging or truncation of the N terminus of M33 resulted in loss of cell surface expression. Within TM II, an F79D mutation abolished constitutive signaling, demonstrating a role, as in other cellular and viral 7TMR, of TM II in receptor activation. In TM III, the arginine (but not the asparagine) residue of the NRY motif (the counterpart of the common DRY motif in cellular 7TMR) was found to be essential for constitutive signaling. Selected mutations incorporated into recombinant MCMV showed that disruption of constitutive signaling for a viral 7TMR homologue resulted in a reduced capacity to disseminate to or replicate in the salivary glands. In addition, HCMV UL33 was found to partially compensate for the lack of M33 in vivo, suggesting conserved biological roles of the UL33 gene family.     

The cytoplasmic end of transmembrane domain 3 regulates the activity of the Saccharomyces cerevisiae G-protein-coupled alpha-factor receptor

The binding of alpha-factor to its receptor (Ste2p) activates a G-protein-signaling pathway leading to conjugation of MATa cells of the budding yeast S. cerevisiae. We conducted a genetic screen to identify constitutively activating mutations in the N-terminal region of the alpha-factor receptor that includes transmembrane domains 1-5. This approach identified 12 unique constitutively activating mutations, the strongest of which affected polar residues at the cytoplasmic ends of transmembrane domains 2 and 3 (Asn84 and Gln149, respectively) that are conserved in the alpha-factor receptors of divergent yeast species. Targeted mutagenesis, in combination with molecular modeling studies, suggested that Gln149 is oriented toward the core of the transmembrane helix bundle where it may be involved in mediating an interaction with Asn84. These residues appear to play specific roles in maintaining the inactive conformation of the protein since a variety of mutations at either position cause constitutive receptor signaling. Interestingly, the activity of many mammalian G-protein-coupled receptors is also regulated by conserved polar residues (the E/DRY motif) at the cytoplasmic end of transmembrane domain 3. Altogether, the results of this study suggest a conserved role for the cytoplasmic end of transmembrane domain 3 in regulating the activity of divergent G-protein-coupled receptors.     

Chimeras of the rat and human FSH receptors (FSHRs) identify residues that permit or suppress transmembrane 6 mutation-induced constitutive activation of the FSHR via rearrangements of hydrophobic interactions between helices 6 and 7

Although a large number of naturally occurring activating mutations of the human LH receptor (hLHR) and human TSH receptor (hTSHR) have been identified, only one activating mutation of the human FSH receptor (hFSHR) has been found. Furthermore, mutations of several residues within the i3/transmembrane domain (TM) 6 region of the hFSHR that were done based upon known constitutively activating mutations of the human LHR were found to have no effect on hFSHR signaling. One of the hFSHR mutations examined in this context was the substitution of a highly conserved aspartate (D581) in TM6 with glycine. We show herein that although the basal activity of the rat FSHR (rFSHR) is similar to the hFSHR, mutation of the comparable residue (D580) in the rFSHR causes marked constitutive activation. Taking advantage of the high degree of amino acid identity between the rat and human FSHRs, we have used chimeras and point substitutions to determine the precise residues that suppress or permit constitutive activity by the D580/581G mutation. Thus, the simultaneous substitution of M576 in TM6 and H615 in TM7 of the hFSHR with the cognate rFSHR residues (threonine and tyrosine, respectively) now renders the hFSHR(D581G) mutant constitutively active. Conversely, the substitution of Y614 of the rFSHR with the cognate hFSHR residue (histidine) fully suppresses the constitutive activity of the rFSHR (D580G) mutant. Computer models of the human and rat FSHRs and mutants thereof were created based upon the crystal structure of rhodopsin. These models suggest that differences in hydrophobic interactions between TMs 6 and 7 of the rat and human FSHRs may account for the ability of TM6 of the rat, but not human, FSHR to adopt an active conformation as a result of the D580/581G mutation.     

Solution structure of the sixth transmembrane helix of the G-protein-coupled receptor, rhodopsin

Low resolution electron density maps have revealed the general orientation of the transmembrane helices of rhodopsin. However, high resolution structural information for the transmembrane domain of the G-protein-coupled receptor, rhodopsin, is as yet unavailable. In this study, a high resolution solution structure is reported for a 15 residue portion of the sixth transmembrane helix of rhodopsin (rhovih) as a free peptide. Helix 6 is one of the transmembrane helices of rhodopsin that contains a proline (amino acid residue 267) and the influence of this proline on the structure of this transmembrane domain was unknown. The structure obtained shows an alpha-helix through most of the sequence. The proline apparently induces only a modest distortion in the helix. Previously, the structure of the intradiskal loop connected to helix 6 was solved. The sequence of this loop contained five residues in common (residues 268-272) with the peptide reported here from the rhovih. The five residues in common between these two structures were superimposed to connect these two structures. The superposition showed a root mean square deviation of 0.2 A. Thus, this five residue sequence formed the same structure in both peptides, indicating that the structure of this region is governed primarily by short range interactions.     

Arabidopsis snc2-1D Activates Receptor-Like Protein-Mediated Immunity Transduced through WRKY70

Plant immune receptors belonging to the receptor-like protein (RLP) family contain extracellular leucine-rich repeats (LRRs) and a short cytoplasmic tail linked by a single transmembrane motif. Here, we report the identification of snc2-1D (for suppressor of npr1-1, constitutive 2), a semidominant Arabidopsis thaliana mutant with constitutively activated defense responses. Map-based cloning of snc2-1D showed that it encodes an RLP. The point mutation in snc2-1D leads to substitution of the second Gly for Arg in the conserved GXXXG motif of the transmembrane helix, suggesting that this residue is important for negative regulation of the protein. Epistasis analysis revealed that the snc2-1D mutant phenotype is not affected by mutations in genes known to be required for the nucleotide binding (NB)-LRR Resistance (R) protein signaling. A suppressor screen of snc2-1D was performed, and map-based cloning of one suppressor revealed that mutations in WRKY70 suppress the constitutive defense responses in snc2-1D, suggesting that WRKY70 functions downstream of snc2-1D. The identification of snc2-1D provides us with a unique system for genetic analysis of resistance pathways downstream of RLPs, which may be distinct from those downstream of NB-LRR type R proteins.     

Involvement of the C-terminal proline-rich motif of G protein-coupled receptor kinases in recognition of activated rhodopsin

G protein-coupled receptor kinases (GRKs) are a family of serine/threonine kinases that phosphorylate many activated G protein-coupled receptors (GPCRs) and play an important role in GPCR desensitization. Our previous work has demonstrated that the C-terminal conserved region (CC) of GRK-2 participates in interaction with rhodopsin and that this interaction is necessary for GRK-2-mediated receptor phosphorylation (Gan, X. Q., Wang, J. Y., Yang, Q. H., Li, Z., Liu, F., Pei, G., and Li, L. (2000) J. Biol. Chem. 275, 8469-8474). In this report, we further investigated whether the CC of other GRKs had the same functions and defined the specific sequences in CC that are required for the functions. The CC regions of GRK-1, GRK-2, and GRK-5, representatives of the three subfamilies of GRKs, could bind rhodopsin in vitro and inhibit GRK-2-mediated phosphorylation of rhodopsin, but not a peptide GRK substrate. Through a series of mutagenesis analyses, a proline-rich motif in the CC was identified as the key element involved in the interaction between the CC region and rhodopsin. Point mutations of this motif not only disrupted the interaction of GRK-2 with rhodopsin but also abolished the ability of GRK-2 to phosphorylate rhodopsin. The findings that the CC region of GRKs interact only with the light-activated but not the non-activated rhodopsin and that the N-terminal domain of GRK-2 interacts with rhodopsin in a light-independent manner suggest that the CC region is responsible for the recognition of activated GPCRs in the canonical model.     

Critical role of transmembrane segment zinc binding in the structure and function of rhodopsin

Zinc deficiency and retinitis pigmentosa are both important factors resulting in retinal dysfunction and night blindness. In this study, we address the critical biochemical and structural relevance of zinc ions in rhodopsin and examine whether zinc deficiency can lead to rhodopsin dysfunction. We report the identification of a high-affinity zinc coordination site within the transmembrane domain of rhodopsin, coordinated by the side chains of two highly conserved residues, Glu(122) in transmembrane helix III and His(211) in transmembrane helix V. We also demonstrate that this zinc coordination is critical for rhodopsin folding, 11-cis-retinal binding, and the stability of the chromophore-receptor interaction, defects of which are observed in retinitis pigmentosa. Furthermore, a cluster of retinitis pigmentosa mutations is localized within and around this zinc binding site. Based on these studies, we believe that improvement in zinc binding to rhodopsin at this site within the transmembrane domain may be a pharmacological approach for the treatment of select retinitis pigmentosa mutations. Transmembrane coordination of zinc may also be an important common principle across G-protein-coupled receptors.     

Construction of a sequence motif characteristic of aminergic G protein-coupled receptors

An approach to discover sequence patterns characteristic of ligand classes is described and applied to aminergic G protein-coupled receptors (GPCRs). Putative ligand-binding residue positions were inferred from considering three lines of evidence: conservation in the subfamily absent or underrepresented in the superfamily, any available mutation data, and the physicochemical properties of the ligand. For aminergic GPCRs, the motif is composed of a conserved aspartic acid in the third transmembrane (TM) domain (rhodopsin position 117) and a conserved tryptophan in the seventh TM domain (rhodopsin position 293); the roles of each are readily justified by molecular modeling of ligand-receptor interactions. This minimally defined motif is an appropriate computational tool for identifying additional, potentially novel aminergic GPCRs from a set of experimentally uncharacterized "orphan" GPCRs, complementing existing sequence matching, clustering, and machine-learning techniques. Motif sensitivity stems from the stepwise addition of residues characteristic of an entire class of ligand (and not tailored for any particular biogenic amine). This sensitivity is balanced by careful consideration of residues (evidence drawn from mutation data, correlation of ligand properties to residue properties, and location with respect to the extracellular face), thereby maintaining specificity for the aminergic class. A number of orphan GPCRs assigned to the aminergic class by this motif were later discovered to be a novel subfamily of trace amine GPCRs, as well as the successful classification of the histamine H4 receptor.     

Activation of the cAMP pathway by the TSH receptor involves switching of the ectodomain from a tethered inverse agonist to an agonist

Several lines of evidence indicate that constraining intramolecular interactions between transmembrane domains are required to maintain G protein-coupled receptors in an inactive conformation in the absence of agonist. For the glycoprotein hormone receptors, which harbor a long amino-terminal ectodomain responsible for hormone binding, it has been suggested that the ectodomain could contribute to these negative constraints. To test this hypothesis, we expressed at the surface of COS-7 cells mutants of the TSH receptor in which variable portions of the amino-terminal ectodomain are replaced by a 19-residue tag from bovine rhodopsin. Whereas none of the rhodopsin-tagged truncated mutants could be activated by saturating concentrations of TSH, the constructs with the shortest amino-terminal extension displayed increased constitutive activity toward the cAMP pathway, when compared with the wild-type holoreceptor. The shortest truncated construct was strongly activated by the introduction of mutations in transmembrane segment VI (D633A), or in the third intracellular loop (A623I) of the receptor. The magnitude of the stimulation was similar to that observed when the same mutations were introduced in the intact wild-type receptor. On the contrary, the shortest truncated construct was unaffected by activating mutations affecting residues of the extracellular loop region (I486F, I568T) or the top of transmembrane segment VII (del658-661). Together, our results are compatible with a model in which activation of the cAMP pathway by the TSH receptor involves switching of the ectodomain from a tethered inverse agonist to a true agonist.     

Requirement of specific intrahelical interactions for stabilizing the inactive conformation of glycoprotein hormone receptors

Systematic analysis of structural changes induced by activating mutations has been frequently utilized to study activation mechanisms of G-protein-coupled receptors (GPCRs). In the thyrotropin receptor and the lutropin receptor (LHR), a large number of naturally occurring mutations leading to constitutive receptor activation were identified. Saturating mutagenesis studies of a highly conserved Asp in the junction of the third intracellular loop and transmembrane domain 6 suggested a participation of this anionic residue in a salt bridge stabilizing the inactive receptor conformation. However, substitution of all conserved cationic residues at the cytoplasmic receptor surface did not support this hypothesis. Asp/Glu residues are a common motif at the N-terminal ends of alpha-helices terminating and stabilizing the helical structure (helix capping). Since Asp/Glu residues in the third intracellular loop/transmembrane domain 6 junction are not only preserved in glycoprotein hormone receptors but also in other GPCRs we speculated that this residue probably participates in an N-terminal helix-capping structure. Poly-Ala stretches are known to form and stabilize alpha-helices. Herein, we show that the function of the highly conserved Asp can be mimicked by poly-Ala substitutions in the LHR and thyrotropin receptor. CD and NMR studies of peptides derived from the juxtamembrane portion of the LHR confirmed the helix extension by the poly-Ala substitution and provided further evidence for an involvement of Asp in a helix-capping structure. Our data implicate that in addition to well established interhelical interactions the inactive conformation of GPCRs is also stabilized by specific intrahelical structures.     

Mutations in the Trp-Ser-X-Trp-Ser motif of the erythropoietin receptor abolish processing, ligand binding, and activation of the receptor.

The erythropoietin receptor (EPOR) is a member of the newly identified cytokine receptor superfamily. A common sequence motif, Trp-Ser-X-Trp-Ser (WSXWS), near the transmembrane domain is highly conserved in this family. To determine the function of this motif, we constructed deletion and insertion mutations in this part of the EPOR and introduced them into an interleukin-3 (IL-3)-dependent hematopoietic Ba/F3 cell line. Cells expressing the wild-type EPOR displayed 1,500 erythropoietin (EPO)-binding sites/cell with a single affinity of about 300 pM and proliferate in the presence of IL-3 or EPO. Ba/F3 cells expressing receptors mutated in the WSXWS motif displayed little EPO binding on the cell surface and did not grow in the presence of EPO. The mutant receptors were retained in the endoplasmic reticulum (ER) and, as such, were unable to bind EPO. A single Gly insertion between the two WS sequences caused defects in receptor structure and function similar to mutations lacking all or part of the WSXWS motif. The EPOR can be activated, resulting in proliferation independent of EPO either by an Arg129 to Cys point mutation or by association with the Friend spleen focus-forming virus (SFFV) envelope glycoprotein gp55. Introduction of the point mutation (Arg129 to Cys) did not activate any of the receptors mutated in the WSXWS motif. Moreover, gp55 did not activate the mutant receptors in Ba/F3 cells. Our study indicates that the WSXWS motif is critical for protein folding, ligand-binding, and signal transduction.