Bin classification of G-protein-coupled peptide receptors

With the use of the binmap method, 154 G-protein-coupled peptide receptors are classified. The binmap coordinates are obtained by using the number of residues between the conserved N residue in TM1 and C in the TM4-TM5 loop, between this C and the conserved P in TM6, and between this P and the last residue of the sequence. The binmap suggests that the cloned fMLP receptor in rabbit belongs in fact to the IL8 receptor type.     

Common structural requirements for heptahelical domain function in class A and class C G protein-coupled receptors

G protein-coupled receptors (GPCRs) are key players in cell communication. Several classes of such receptors have been identified. Although all GPCRs possess a heptahelical domain directly activating G proteins, important structural and sequence differences within receptors from different classes suggested distinct activation mechanisms. Here we show that highly conserved charged residues likely involved in an interaction network between transmembrane domains (TM) 3 and 6 at the cytoplasmic side of class C GPCRs are critical for activation of the gamma-aminobutyric acid type B receptor. Indeed, the loss of function resulting from the mutation of the conserved lysine residue into aspartate or glutamate in the TM3 of gamma-aminobutyric acid type B(2) can be partly rescued by mutating the conserved acidic residue of TM6 into either lysine or arginine. In addition, mutation of the conserved lysine into an acidic residue leads to a nonfunctional receptor that displays a high agonist affinity. This is reminiscent of a similar ionic network that constitutes a lock stabilizing the inactive state of many class A rhodopsin-like GPCRs. These data reveal that despite their original structure, class C GPCRs share with class A receptors at least some common structural feature controlling G protein activation.     

A conserved Asn in transmembrane helix 7 is an on/off switch in the activation of the thyrotropin receptor

The thyrotropin (TSH) receptor is an interesting model to study G protein-coupled receptor activation as many point mutations can significantly increase its basal activity. Here, we identified a molecular interaction between Asp(633) in transmembrane helix 6 (TM6) and Asn(674) in TM7 of the TSHr that is crucial to maintain the inactive state through conformational constraint of the Asn. We show that these residues are perfectly conserved in the glycohormone receptor family, except in one case, where they are exchanged, suggesting a direct interaction. Molecular modeling of the TSHr, based on the high resolution structure of rhodopsin, strongly favors this hypothesis. Our approach combining site-directed mutagenesis with molecular modeling shows that mutations disrupting this interaction, like the D633A mutation in TM6, lead to high constitutive activation. The strongly activating N674D (TM7) mutation, which in our modeling breaks the TM6-TM7 link, is reverted to wild type-like behavior by an additional D633N mutation (TM6), which would restore this link. Moreover, we show that the Asn of TM7 (conserved in most G protein-coupled receptors) is mandatory for ligand-induced cAMP accumulation, suggesting an active role of this residue in activation. In the TSHr, the conformation of this Asn residue of TM7 would be constrained, in the inactive state, by its Asp partner in TM6.     

Participation of transmembrane proline 82 in angiotensin II AT1 receptor signal transduction

Most of the classical physiological effects of the octapeptide angiotensin II (AngII) are produced by activating the AT1 receptor which belongs to the G-protein coupled receptor family (GPCR). Peptidic GPCRs may be functionally divided in three regions: (i) extracellular domains involved in ligand binding; (ii) intracellular domains implicated in agonist-induced coupling to G protein and (iii) seven transmembrane domains (TM) involved in signal transduction. The TM regions of such receptors have peculiar characteristics such as the presence of proline residues. In this project we aimed to investigate the participation of two highly conserved proline residues (Pro82 and Pro162), located in TM II and TM IV, respectively, in AT1 receptor signal transduction. Both mutations did not cause major alterations in AngII affinity. Functional assays indicated that the P162A mutant did not influence the signal transduction. On the other hand, a potent deleterious effect of P82A mutation on signal transduction was observed. We believe that the Pro82 residue is crucial to signal transduction, although it is not possible to say yet if this is due to a direct participation or if due to a structural rearrangement of TM II. In this last hypothesis, the removal of proline residue might be correlated to a removal of a kink, which in turn can be involved in the correct positioning of residues involved in signal transduction.     

Highly conserved tyrosine 37 stabilizes desensitized states and restricts calcium permeability of ATP-gated P2X3 receptor

Tyrosine 37 in the first transmembrane (TM1) domain is highly conserved in ATP-gated P2X receptors suggesting its fundamental role. We tested whether Y37 contributes to the desensitization of P2X3 receptors, which is currently not well understood. By combining electrophysiological, imaging and modeling approaches, we studied desensitization of various Y37 P2X3 mutants and potential partners of Y37. Unlike the membrane current of the WT receptor, which desensitized in seconds, Y37A mutant current did not fully desensitize even after minutes-long applications of β,γ-meATP, α,β-meATP, ATP or 2MeS-ATP. The fractional calcium current was enhanced in the Y37A mutant. Y37F did not rescue the native P2X3 phenotype indicating a role for the hydroxyl group of Y37 for the WT receptor. Homology modeling indicated I318 or I319 in TM2 as potential partners for Y37 in the receptor closed state. We tested this hypothesis by creating a permanent interaction between the two residues via disulfide bond. Whereas single Y37C, I318C and I319C mutants were functional, the double mutants Y37C-I318C and Y37C-I319C were non-functional. Using a cyclic model of receptor operation, we suggest that the conserved tyrosine 37 links TM1 to TM2 of adjacent subunit to stabilize desensitized states and restricts calcium permeability through the ion channel.     

Structural Insights into the Function of P2X4: An ATP-Gated Cation Channel of Neuroendocrine Cells

The P2X4 receptor (P2X4R) is a member of a family of ATP-gated cation channels that are composed of three subunits. Each subunit has two transmembrane (TM) domains linked by a large extracellular loop and intracellularly located N- and C-termini. The receptors are expressed in excitable and non-excitable cells and have been implicated in the modulation of membrane excitability, calcium signaling, neurotransmitter and hormone release, and pain physiology. P2X4Rs activate rapidly and desensitize within the seconds of agonist application, both with the rates dependent on ATP concentrations, and deactivate rapidly and independently of ATP concentration. Disruption of conserved cysteine ectodomain residues affects ATP binding and gating. Several ectodomain residues of P2X4R were identified as critical for ATP binding, including K67, K313, and R295. Ectodomain residues also account for the allosteric regulation of P2X4R; H140 is responsible for copper binding and H286 regulates receptor functions with protons. Ivermectin sensitized receptors, amplified the current amplitude, and slowed receptor deactivation by binding in the TM region. Scanning mutagenesis of TMs revealed the helical topology of both domains, and suggested that receptor function is critically dependent on the conserved Y42 residue. In this brief article, we summarize this study and re-interpret it using a model based on crystallization of the zebrafish P2X4.1 receptor.     

Expression and function of the gonadotropin-releasing hormone receptor are dependent on a conserved apolar amino acid in the third intracellular loop

The coupling of agonist-activated heptahelical receptors to their cognate G proteins is often dependent on the amino-terminal region of the third intracellular loop. Like many G protein-coupled receptors, the gonadotropin-releasing hormone (GnRH) receptor contains an apolar amino acid in this region at a constant distance from conserved Pro and Tyr/Asn residues in the fifth transmembrane domain (TM V). An analysis of the role of this conserved residue (Leu(237)) in GnRH receptor function revealed that the binding affinities of the L237I and L237V mutant receptors were unchanged, but their abilities to mediate GnRH-induced inositol phosphate signaling, G protein coupling, and agonist-induced internalization were significantly impaired. Receptor expression at the cell surface was reduced by replacement of Leu(237) with Val, and abolished by replacement with Ala, Arg, or Asp residues. These results are consistent with molecular modeling of the TM V and VI regions of the GnRH receptor, which predicts that Leu(237) is caged by several apolar amino acids (Ile(233), Ile(234), and Val(240) in TM V, and Leu(262), Leu(265), and Val(269) in TM VI) to form a tight hydrophobic cluster. These findings indicate that the conserved apolar residue (Leu(237)) in the third intracellular loop is an important determinant of GnRH receptor expression and activation, and possibly that of other G protein-coupled receptors.     

A region in the seven-transmembrane domain of the human Ca2+ receptor critical for response to Ca2+

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.     

Direct Gating of ATP-activated Ion Channels (P2X2 Receptors) by Lipophilic Attachment at the Outer End of the Second Transmembrane Domain

The ionic pore of the P2X receptor passes through the central axis of six transmembrane (TM) helices, two from each of three subunits. Val⁴⁸ and Ile³²⁸ are at the outer end of TM1 and TM2, respectively. Homology models of the open and closed states of P2X2 indicate that pore opening is associated with a large lateral displacement of Ile³²⁸. In addition, molecular dynamics simulations suggest that lipids enter the interstices between the outer ends of the TM domains. The P2X2(I328C) receptor was activated by propyl-methanethiosulfonate (MTS) as effectively as by ATP, but cysteine substitutions elsewhere in TM2 had no such effect. Other lipophilic MTS compounds (methyl, ethyl, and tert-butylethyl) had a similar effect but not polar MTS. The properties of the conducting pathway opened by covalent attachment of propyl-MTS were the same as those opened by ATP, with respect to unitary conductance, rectification, and permeability of N-methyl-d-glucamine. The ATP-binding residue Lys⁶⁹ was not required for the action of propyl-MTS, although propyl-MTS did not open P2X2(K308A/I328C) receptors. The propyl-MTS did not open P2X2 receptors in which the Val⁴⁸ side chain was removed (P2X2(V48G/I328C)). The results suggest that an interaction between Val⁴⁸ and Ile³²⁸ stabilizes the closed channel and that this is broken by covalent attachment of a larger lipophilic moiety at the I328C receptors. Lipid intercalation between the separating TM domains during channel opening would be facilitated in P2X2(I328C) receptors with attached propyl-MTS. The results are consistent with the channel opening mechanism proposed on the basis of closed and open crystal structures and permit the refinement of the position of the TMs within the bilayer.     

The first transmembrane segment of the dopamine D2 receptor: accessibility in the binding-site crevice and position in the transmembrane bundle

The binding site of the dopamine D2 receptor, like that of homologous G-protein-coupled receptors (GPCRs), is contained within a water-accessible crevice formed among its seven transmembrane segments (TMs). Using the substituted-cysteine-accessibility method (SCAM), we are mapping the residues that contribute to the surface of this binding-site crevice. We have now mutated to cysteine, one at a time, 21 consecutive residues in TM1. Six of these mutants reacted with charged sulfhydryl reagents, whereas bound antagonist only protected N52(1.50)C from reaction. Except for A38(1.36)C, none of the substituted cysteine mutants in the extracellular half of TM1 appeared to be accessible. Pro(1.48) is highly conserved in opsins, but absent in catecholamine receptors, and the high-resolution rhodopsin structure showed that Pro(1.48) bends the extracellular portion of TM1 inward toward TM2 and TM7. Analysis of the conversation of residues in the extracellular portion of TM1 of opsins showed a pattern consistent with alpha-helical structure with a conserved face. In contrast, this region in catecholamine receptors is poorly conserved, suggesting a lack of critical contacts. Thus, in catecholamine receptors in the absence of Pro(1.48), TM1 may be straighter and therefore further from the helix bundle, consistent with the apparent lack of conserved contact residues. When examined in the context of a model of the D2 receptor, the accessible residues in the cytoplasmic half of TM1 are at the interface with TM7 and with helix 8 (H8). We propose the existence of critical contacts of TM1, TM7, and H8 that may stabilize the inactive state of the receptor.     

An activation switch in the rhodopsin family of G protein-coupled receptors: the thyrotropin receptor

We aimed at understanding molecular events involved in the activation of a member of the G protein-coupled receptor family, the thyrotropin receptor. We have focused on the transmembrane region and in particular on a network of polar interactions between highly conserved residues. Using molecular dynamics simulations and site-directed mutagenesis techniques we have identified residue Asn-7.49, of the NPxxY motif of TM 7, as a molecular switch in the mechanism of thyrotropin receptor (TSHr) activation. Asn-7.49 appears to adopt two different conformations in the inactive and active states. These two states are characterized by specific interactions between this Asn and polar residues in the transmembrane domain. The inactive gauche+ conformation is maintained by interactions with residues Thr-6.43 and Asp-6.44. Mutation of these residues into Ala increases the constitutive activity of the receptor by factors of approximately 14 and approximately 10 relative to wild type TSHr, respectively. Upon receptor activation Asn-7.49 adopts the trans conformation to interact with Asp-2.50 and a putatively charged residue that remains to be identified. In addition, the conserved Leu-2.46 of the (N/S)LxxxD motif also plays a significant role in restraining the receptor in the inactive state because the L2.46A mutation increases constitutive activity by a factor of approximately 13 relative to wild type TSHr. As residues Leu-2.46, Asp-2.50, and Asn-7.49 are strongly conserved, this molecular mechanism of TSHr activation can be extended to other members of the rhodopsin-like family of G protein-coupled receptors.     

PheVI:09 (Phe6.44) as a Sliding Microswitch in Seven-transmembrane (7TM) G Protein-coupled Receptor Activation

In seven-transmembrane (7TM), G protein-coupled receptors, highly conserved residues function as microswitches, which alternate between different conformations and interaction partners in an extended allosteric interface between the transmembrane segments performing the large scale conformational changes upon receptor activation. Computational analysis using x-ray structures of the β₂-adrenergic receptor demonstrated that PheVI:09 (6.44), which in the inactive state is locked between the backbone and two hydrophobic residues in transmembrane (TM)-III, upon activation slides ∼2 Å toward TM-V into a tight pocket generated by five hydrophobic residues protruding from TM-III and TM-V. Of these, the residue in position III:16 (3.40) (often an Ile or Val) appears to function as a barrier or gate for the transition between inactive and active conformation. Mutational analysis showed that PheVI:09 is essential for the constitutive and/or agonist-induced signaling of the ghrelin receptor, GPR119, the β₂-adrenergic receptor, and the neurokinin-1 receptor. Substitution of the residues constituting the hydrophobic pocket between TM-III and TM-V in the ghrelin receptor in four of five positions impaired receptor signaling. In GPR39, representing the 12% of 7TM receptors lacking an aromatic residue at position VI:09, unchanged agonist-induced signaling was observed upon Ala substitution of LeuVI:09 despite reduced cell surface expression of the mutant receptor. It is concluded that PheVI:09 constitutes an aromatic microswitch that stabilizes the active, outward tilted conformation of TM-VI relative to TM-III by sliding into a tight hydrophobic pocket between TM-III and TM-V and that the hydrophobic residue in position III:16 constitutes a gate for this transition.     

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.     

Helix 8 of the viral chemokine receptor ORF74 directs chemokine binding

The constitutively active G-protein-coupled receptor and viral oncogene ORF74, encoded by Kaposi sarcoma-associated herpesvirus (human herpesvirus 8), binds a broad range of chemokines, including CXCL1 (agonist), CXCL8 (neutral ligand), and CXCL10 (inverse agonist). Although chemokines interact with the extracellular N terminus and loops of the receptor, we demonstrate that helix 8 (Hx8) in the intracellular carboxyl tail (C-tail) of ORF74 directs chemokine binding. Partial deletion of the C-tail resulted in a phenotype with reduced constitutive activity but intact regulation by ligands. Complete deletion of the C-tail, including Hx8, resulted in an inactive phenotype that lacks CXCL8 binding sites and has an increased number of binding sites for CXCL10. Similar effects were obtained with the single R7.61(322)W or Q7.62(323)P mutations in Hx8. We propose that the conserved charged or polar side chain at position 7.61 has a specific role in stabilizing the end of transmembrane domain 7 (TM7). Disruption of Hx8 by deletion or mutation distorts an H-bonding network, involving highly conserved amino acids within TM2, TM7, and Hx8, that is crucial for positioning of the TM domains, coupling to Galphaq, and CXCL8 binding. Thus, Hx8 appears to exert a key role in receptor stabilization through the conserved residue R7.61, directing the ligand binding profile of ORF74 and likely also that of other class A G-protein-coupled receptors.     

Fv-4: identification of the defect in Env and the mechanism of resistance to ecotropic murine leukemia virus

Mice expressing the Fv-4 gene are resistant to infection by ecotropic murine leukemia viruses (MuLVs). The Fv-4 gene encodes an envelope (Env) protein whose putative receptor-binding domain resembles that of ecotropic MuLV Env protein. Resistance to ecotropic MuLVs appears to result from viral interference involving binding of the endogenously expressed Fv-4 env-encoded protein to the ecotropic receptor, although the immune system also plays a role in resistance. The Fv-4 env-encoded protein is processed normally and can be incorporated into virus particles but is unable to promote viral entry. Among the many sequence variations between the transmembrane (TM) subunit of the Fv-4 env-encoded protein and the TM subunits of other MuLV Env proteins, there is a substitution of an arginine residue in the Fv-4 env-encoded protein for a glycine residue (gly-491 in Moloney MuLV Env) that is otherwise conserved in all of the other MuLVs. This residue is present in the MuLV TM fusion peptide sequence. In this study, gly-491 of Moloney MuLV Env has been replaced with other residues and a mutant Env bearing a substitution for gly-487 was also created. G491R recapitulates the Fv-4 Env phenotype in cell culture, indicating that this substitution is sufficient for creation of an Env protein that can establish the interference-mediated resistance to ecotropic viruses produced by the Fv-4 gene. Analysis of the mutant MuLV Env proteins also has implications for an understanding of the role of conserved glycine residues in fusion peptides and for the engineering of organismal resistance to retroviruses.     

Charged extracellular residues, conserved throughout a G-protein-coupled receptor family, are required for ligand binding, receptor activation, and cell-surface expression

For G-protein-coupled receptors (GPCRs) in general, the roles of extracellular residues are not well defined compared with residues in transmembrane helices (TMs). Nevertheless, extracellular residues are important for various functions in both peptide-GPCRs and amine-GPCRs. In this study, the V(1a) vasopressin receptor was used to systematically investigate the role of extracellular charged residues that are highly conserved throughout a subfamily of peptide-GPCRs, using a combination of mutagenesis and molecular modeling. Of the 13 conserved charged residues identified in the extracellular loops (ECLs), Arg(116) (ECL1), Arg(125) (top of TMIII), and Asp(204) (ECL2) are important for agonist binding and/or receptor activation. Molecular modeling revealed that Arg(125) (and Lys(125)) stabilizes TMIII by interacting with lipid head groups. Charge reversal (Asp(125)) caused re-ordering of the lipids, altered helical packing, and increased solvent penetration of the TM bundle. Interestingly, a negative charge is excluded at this locus in peptide-GPCRs, whereas a positive charge is excluded in amine-GPCRs. This contrasting conserved charge may reflect differences in GPCR binding modes between peptides and amines, with amines needing to access a binding site crevice within the receptor TM bundle, whereas the binding site of peptide-GPCRs includes more extracellular domains. A conserved negative charge at residue 204 (ECL2), juxtaposed to the highly conserved disulfide bond, was essential for agonist binding and signaling. Asp(204) (and Glu(204)) establishes TMIII contacts required for maintaining the beta-hairpin fold of ECL2, which if broken (Ala(204) or Arg(204)) resulted in ECL2 unfolding and receptor dysfunction. This study provides mechanistic insight into the roles of conserved extracellular residues.     

Asn229 in the third helix of VPAC1 receptor is essential for receptor activation but not for receptor phosphorylation and internalization: comparison with Asn216 in VPAC2 receptor

After stimulation with agonist, G protein coupled receptors (GPCR) undergo conformational changes that allow activation of G proteins to transduce the signal, followed by phosphorylation by kinases and arrestin binding to promote receptor internalization. Actual paradigm, based on a study of GPCR-A/rhodopsin family, suggests that a network of interactions between conserved residues located in transmembrane (TM) domains (mainly TM3, TM6 and TM7) is involved in the molecular switch leading to GPCR activation.

We evaluated in CHO cells expressing the VPAC₁ receptor the role of the third transmembrane helix in agonist signalling by point mutation into Ala of the residues highly conserved in the secretin-family of receptors: Y²²⁴, N²²⁹, F²³⁰, W²³², E²³⁶, G²³⁷, Y²³⁹, L²⁴⁰. N²²⁹A VPAC₁ mutant was characterized by a decrease in both potency and efficacy of VIP stimulated adenylate cyclase activity, by the absence of agonist stimulated [Ca²⁺]_i increase, by a preserved receptor recognition of agonists and antagonist and by a preserved sensitivity to GTP suggesting the importance of that residue for efficient G protein activation. N²²⁹D mutant was not expressed at the membrane, and the N²²⁹Q with a conserved mutation was less affected than the A mutant. Agonist stimulated phosphorylation and internalization of N²²⁹A and N²²⁹Q VPAC₁ were unaffected. However, the re-expression of internalized mutant receptors, but not that of the wild type receptor, was rapidly reversed after VIP washing. Receptor phosphorylation, internalization and re-expression may be thus dissociated from G protein activation and linked to another active conformation that may influence its trafficking.

Mutation of that conserved amino acid in VPAC₂ could be investigated only by a conservative mutation (N²¹⁶Q) and led to a receptor with a low VIP stimulation of adenylate cyclase, receptor phosphorylation and internalization. This indicated the importance of the conserved N residue in the TM3 of that family of receptors.     

Third Extracellular Loop (EC3)-N Terminus Interaction Is Important for Seven-transmembrane Domain Receptor Function: IMPLICATIONS FOR AN ACTIVATION MICROSWITCH REGION*

The canonical heptahelical bundle architecture of seven-transmembrane domain (7TM) receptors is intertwined by three intra- and three extracellular loops, whose local conformations are important in receptor signaling. Many 7TM receptors contain a cysteine residue in the third extracellular loop (EC3) and a complementary cysteine residue on the N terminus. The functional role of such EC3-N terminus conserved cysteine pairs remains unclear. This study explores the role of the EC3-N terminus cysteine pairs on receptor conformation and G protein activation by disrupting them in the chemokine receptor CXCR4, while engineering a novel EC3-N terminus cysteine pair into the complement factor 5a receptor (C5aR), a chemo attractant receptor that lacks it. Mutated CXCR4 and C5aRs were expressed in engineered yeast. Mutation of the cysteine pair with the serine pair (C28S/C274S) in constitutively active mutant CXCR4 abrogated the receptor activation, whereas mutation with the aromatic pair (C28F–C274F) or the salt bridge pair (C28R/C274E), respectively, rescued or retained the receptor activation in response to CXCL12. In this context, the cysteine pair (Cys³⁰ and Cys²⁷²) engineered into the EC3-N terminus (Ser³⁰ and Ser²⁷²) of a novel constitutively active mutant of C5aR restrained the constitutive signaling without affecting the C5a-induced activation. Further mutational studies demonstrated a previously unappreciated role for Ser²⁷² on EC3 of C5aR and its interaction with the N terminus, thus defining a new microswitch region within the C5aR. Similar results were obtained with mutated CXCR4 and C5aRs expressed in COS-7 cells. These studies demonstrate a novel role of the EC3-N terminus cysteine pairs in G protein-coupled receptor activation and signaling.     

Random mutagenesis of the M3 muscarinic acetylcholine receptor expressed in yeast: identification of second-site mutations that restore function to a coupling-deficient mutant M3 receptor

The M(3) muscarinic receptor is a prototypical member of the class A family of G protein-coupled receptors (GPCRs). To gain insight into the structural mechanisms governing agonist-mediated M(3) receptor activation, we recently developed a genetically modified yeast strain (Saccharomyces cerevisiae) which allows the efficient screening of large libraries of mutant M(3) receptors to identify mutant receptors with altered/novel functional properties. Class A GPCRs contain a highly conserved Asp residue located in transmembrane domain II (TM II; corresponding to Asp-113 in the rat M(3) muscarinic receptor) which is of fundamental importance for receptor activation. As observed previously with other GPCRs analyzed in mammalian expression systems, the D113N point mutation abolished agonist-induced receptor/G protein coupling in yeast. We then subjected the D113N mutant M(3) receptor to PCR-based random mutagenesis followed by a yeast genetic screen to recover point mutations that can restore G protein coupling to the D113N mutant receptor. A large scale screening effort led to the identification of three such second-site suppressor mutations, R165W, R165M, and Y250D. When expressed in the wild-type receptor background, these three point mutations did not lead to an increase in basal activity and reduced the efficiency of receptor/G protein coupling. Similar results were obtained when the various mutant receptors were expressed and analyzed in transfected mammalian cells (COS-7 cells). Interestingly, like Asp-113, Arg-165 and Tyr-250, which are located at the cytoplasmic ends of TM III and TM V, respectively, are also highly conserved among class A GPCRs. Our data suggest a conformational link between the highly conserved Asp-113, Arg-165, and Tyr-250 residues which is critical for receptor activation.     

Functional response of the small multidrug resistance protein EmrE to mutations in transmembrane helix 2

Escherichia coli EmrE is a small multidrug resistance protein encompassing four transmembrane (TM) sequences that oligomerizes to confer resistance to antimicrobials. Here we examined the effects on in vivo protein accumulation and ethidium resistance activity of single residue substitutions at conserved and variable positions in EmrE transmembrane segment 2 (TM2). We found that activity was reduced when conserved residues localized to one TM2 surface were replaced. Our findings suggest that conserved TM2 positions tolerate greater residue diversity than conserved sites in other EmrE TM sequences, potentially reflecting a source of substrate polyspecificity.