The role of conserved extracellular cysteine residues in vasopressin V2 receptor function and properties of two naturally occurring mutant receptors with additional extracellular cysteine residues

The G protein-coupled vasopressin V2 receptor (V2 receptor) contains a pair of conserved cysteine residues (C112 and C192) which are thought to form a disulfide bond between the first and second extracellular loops. The conserved cysteine residues were found to be important for the correct formation of the ligand binding domain of some G protein-coupled receptors. Here we have assessed the properties of the V2 receptor after site-directed mutagenesis of its conserved cysteine residues in transiently transfected human embryonic kidney (HEK 293) cells. Mutant receptors (C112S, C112A and C192S, C192A) were non-functional and located mostly in the cell's interior. The conserved cysteine residues of the V2 receptor are thus not only important for the structure of the ligand binding domain but also for efficient intracellular receptor transport. In addition to the functional significance of the conserved cysteine residues, we have also analyzed the defects of two mutant V2 receptors which cause X-linked nephrogenic diabetes insipidus (NDI) by the introduction of additional cysteine residues into the second extracellular loop (mutants G185C, R202C). These mutations are assumed to impair normal disulfide bond formation. Mutant receptor G185C and R202C were efficiently transported to the plasma membrane but were defective in ligand binding. Only in the case of the mutant receptor R202C, the more sensitive adenylyl cyclase activity assay revealed vasopressin-stimulated cAMP formation with a 35-fold increased EC(50) value and with a reduced EC(max), indicating that ligand binding is not completely abolished. Taking the unaffected intracellular transport of both NDI-causing mutant receptors into account, our results indicate that the observed impairment of ligand binding by the additional cysteine residues is not due to the prevention of disulfide bond formation between the conserved cysteine residues.     

Conserved Extracellular Cysteine Pair in the M3 Muscarinic Acetylcholine Receptor Is Essential for Proper Receptor Cell Surface Localization but Not for G Protein Coupling

Most G protein-coupled receptors contain a conserved pair of extracellular cysteine residues that are predicted to form a disulfide bond linking the first and second extracellular loops. Previous studies have shown that this disulfide bond may be critical for ligand binding, receptor activation, and/or proper receptor folding. However, the potential importance of the two conserved cysteine residues for proper receptor cell surface localization has not been investigated systematically. To address this issue, we used the rat M3 muscarinic receptor as a model system. Most studies were carried out with a modified version of this receptor subtype (lacking potential N-glycosylation sites and the central portion of the third intracellular loop) that could be readily detected via western blot analysis. Cys-->Ala mutant receptors were generated, transiently expressed in COS-7 cells, and then examined for their subcellular distribution and functional properties. ELISA and immunofluorescence studies showed that the presence of both conserved cysteine residues (corresponding to C140 and C220 in the rat M3 muscarinic receptor sequence) is required for efficient expression of the M3 muscarinic receptor on the cell surface. On the other hand, these residues were found not to be essential for protein stability (determined via immunoblotting) and receptor-mediated G protein activation (studied in second messenger assays). These results shed new light on the functional role of the two extracellular cysteine residues present in most G protein-coupled receptors.     

Paired cysteine mutagenesis to establish the pattern of disulfide bonds in the functional intact secretin receptor

The amino-terminal domain of class B G protein-coupled receptors contains six conserved cysteine residues involved in structurally and functionally critical disulfide bonds. The mapping of these bonds has been unclear, with one pattern based on biochemical and NMR structural characterizations of refolded, nonglycosylated amino-terminal fragments, and another pattern derived from functional characterizations of intact receptors having paired cysteine mutations. In the present study, we determined the disulfide bonding pattern of the prototypic class B secretin receptor by applying the same paired cysteine mutagenesis approach and confirming the predicted bonding pattern with proteolytic cleavage of intact functional receptor. As expected, systematic mutation to serine of the six conserved cysteine residues within this region of the secretin receptor singly and in pairs resulted in loss of function of most constructs. Notable exceptions were single mutations of the 4th and 6th cysteine residues and paired mutations involving the 1st and 3rd, 2nd and 5th, and 4th and 6th conserved cysteines, with secretin eliciting statistically significant cAMP responses above basal levels of activation for each of these constructs. Immunofluorescence microscopy confirmed similar levels of plasma membrane expression for each of the mutated receptors. Furthermore, cyanogen bromide cleaved a series of wild type and mutant secretin receptors, yielding patterns that agreed with our paired cysteine mutagenesis results. In conclusion, these data suggest the same pattern of disulfide bonding as that predicted previously by NMR and thus support a consistent pattern of amino-terminal disulfide bonds in class B G protein-coupled receptors.     

Use of a disulfide cross-linking strategy to study muscarinic receptor structure and mechanisms of activation.

To gain insight into the molecular architecture of the cytoplasmic surface of G protein-coupled receptors, we have developed a disulfide cross-linking strategy using the m3 muscarinic receptor as a model system. To facilitate the interpretation of disulfide cross-linking data, we initially generated a mutant m3 muscarinic receptor (referred to as m3'(3C)-Xa) in which most native Cys residues had been deleted or substituted with Ala or Ser (remaining Cys residues Cys-140, Cys-220, and Cys-532) and in which the central portion of the third intracellular loop had been replaced with a factor Xa cleavage site. Radioligand binding and second messenger assays showed that the m3'(3C)-Xa mutant receptor was fully functional. In the next step, pairs of Cys residues were reintroduced into the m3'(3C)-Xa construct, thus generating 10 double Cys mutant receptors. All 10 mutant receptors contained a Cys residue at position 169 at the beginning of the second intracellular loop and a second Cys within the C-terminal portion of the third intracellular loop, at positions 484-493. Radioligand binding studies and phosphatidylinositol assays indicated that all double Cys mutant receptors were properly folded. Membrane lysates prepared from COS-7 cells transfected with the different mutant receptor constructs were incubated with factor Xa protease and the oxidizing agent Cu(II)-(1,10-phenanthroline)3, and the formation of intramolecular disulfide bonds between juxtaposed Cys residues was monitored by using a combined immunoprecipitation/immunoblotting strategy. To our surprise, efficient disulfide cross-linking was observed with 8 of the 10 double Cys mutant receptors studied (Cys-169/Cys-484 to Cys-491), suggesting that the intracellular m3 receptor surface is characterized by pronounced backbone fluctuations. Moreover, [35S]guanosine 5'-3-O-(thio)triphosphate binding assays indicated that the formation of intramolecular disulfide cross-links prevented or strongly inhibited receptor-mediated G protein activation, suggesting that the highly dynamic character of the cytoplasmic receptor surface is a prerequisite for efficient receptor-G protein interactions. This is the first study using a disulfide mapping strategy to examine the three-dimensional structure of a hormone-activated G protein-coupled receptor.     

Mutating the four extracellular cysteines in the chemokine receptor CCR6 reveals their differing roles in receptor trafficking,ligand binding,and signaling

CCR6 is the receptor for the chemokine MIP-3 alpha/CCL20. Almost all chemokine receptors contain cysteine residues in the N-terminal domain and in the first, second, and third extracellular loops. In this report, we have studied the importance of all cysteine residues in the CCR6 sequence using site-directed mutagenesis and biochemical techniques. Like all G protein-coupled receptors, mutating disulfide bond-forming cysteines in the first (Cys118) and second (Cys197) extracellular loops in CCR6 led to complete elimination of receptor activity, which for CCR6 was also associated with the accumulation of the receptor intracellularly. Although two additional cysteines in the N-terminal region and the third extracellular loop, which are present in almost all chemokine receptors, are presumed to form a disulfide bond, this has not been demonstrated experimentally for any of these receptors. We found that mutating the cysteines in the N-terminal domain (Cys36) and the third extracellular loop (Cys288) neither significantly affected receptor surface expression nor completely abolished receptor function. Importantly, contrary to several previous reports, we demonstrated directly that instead of forming a disulfide bond, the N-terminal cysteine (Cys36) and the third extracellular loop cysteine (Cys288) contain free SH groups. The cysteine residues (Cys36 and Cys288), rather than forming a disulfide bond, may be important per se. We propose that CCR6 forms only a disulfide bond between the first (Cys118) and second (Cys197) extracellular loops, which confines a helical bundle together with the N-terminus adjacent to the third extracellular loop, creating the structural organization critical for ligand binding and therefore for receptor signaling.     

Essential role for the second extracellular loop in C5a receptor activation

More than 90% of G protein-coupled receptors (GPCRs) contain a disulfide bridge that tethers the second extracellular loop (EC2) to the third transmembrane helix. To determine the importance of EC2 and its disulfide bridge in receptor activation, we subjected this region of the complement factor 5a receptor (C5aR) to random saturation mutagenesis and screened for functional receptors in yeast. The cysteine forming the disulfide bridge was the only conserved residue in the EC2-mutated receptors. Notably, approximately 80% of the functional receptors exhibited potent constitutive activity. These results demonstrate an unexpected role for EC2 as a negative regulator of C5a receptor activation. We propose that in other GPCRs, EC2 might serve a similar role by stabilizing the inactive state of the receptor.     

Reversible inactivation of AT(2) angiotensin II receptor from cysteine-disulfide bond exchange

Dithiothreitol (DTT) treatment of angiotensin II (Ang II) type 2 (AT(2)) receptor potentiates ligand binding, but the underlying mechanism is not known. Two disulfide bonds proposed in the extracellular domain were examined in this report. Based on the analysis of ligand affinity of cysteine (Cys, C) to alanine (Ala, A) substitution mutants, we provide evidence that Cys(35)-Cys(290) and Cys(117)-Cys(195) disulfide bonds are formed in the wild-type AT(2) receptor. Disruption of the highly conserved Cys(117)-Cys(195) disulfide bond linking the second and third extracellular segments leads to inactivation of the receptor. The Cys(35)-Cys(290) bond is highly sensitive to DTT. Its breakage results in an increased binding affinity for both Ang II and the AT(2) receptor-specific antagonist PD123319. Surprisingly, in the single Cys mutants, C35A and C290A, a labile population of receptors is produced which can be re-folded to high-affinity state by DTT treatment. These results suggest that the free -SH group of Cys(35) or Cys(290) competes with the disulfide bond formation between Cys(117) and Cys(195). This Cys-disulfide bond exchange results in production of the inactive population of the mutant receptors through formation of a non-native disulfide bond.     

Disulfide bond configuration of human cytomegalovirus glycoprotein B

Glycoprotein B (gB) is the most highly conserved of the envelope glycoproteins of human herpesviruses. The gB protein of human cytomegalovirus (CMV) serves multiple roles in the life cycle of the virus. To investigate structural properties of gB that give rise to its function, we sought to determine the disulfide bond arrangement of gB. To this end, a recombinant form of gB (gB-S) comprising the entire ectodomain of the glycoprotein (amino acids 1 to 750) was constructed and expressed in insect cells. Proteolytic fragmentation and mass spectrometry were performed using purified gB-S, and the five disulfide bonds that link 10 of the 11 highly conserved cysteine residues of gB were mapped. These bonds are C94-C550, C111-C506, C246-C250, C344-C391, and C573-C610. This configuration closely parallels the disulfide bond configuration of herpes simplex type 2 (HSV-2) gB (N. Norais, D. Tang, S. Kaur, S. H. Chamberlain, F. R. Masiarz, R. L. Burke, and F. Markus, J. Virol. 70:7379-7387, 1996). However, despite the high degree of conservation of cysteine residues between CMV gB and HSV-2 gB, the disulfide bond arrangements of the two homologs are not identical. We detected a disulfide bond between the conserved cysteine residue 246 and the nonconserved cysteine residue 250 of CMV gB. We hypothesize that this disulfide bond stabilizes a tight loop in the amino-terminal fragment of CMV gB that does not exist in HSV-2 gB. We predicted that the cysteine residue not found in a disulfide bond of CMV gB, cysteine residue 185, would play a role in dimerization, but a cysteine substitution mutant in cysteine residue 185 showed no apparent defect in the ability to form dimers. These results indicate that gB oligomerization involves additional interactions other than a single disulfide bond. This work represents the second reported disulfide bond structure for a herpesvirus gB homolog, and the discovery that the two structures are not identical underscores the importance of empirically determining structures even for highly conserved proteins.     

Identification and function of disulfide bridges in the extracellular domains of the angiotensin II type 2 receptor.

The angiotensin II (AngII) receptor family is comprised of two subtypes, type 1 (AT(1)) and type 2 (AT(2)). Although sharing low homology (only 34%), mutagenesis has identified some key residues that are conserved between both subtypes, including four extracellular cysteines. Previous AT(1) mutagenesis demonstrated that the cysteines form two disulfide bonds, one linking the first and second extracellular loops and another connecting the amino terminus to the third extracellular loop. The importance of these AT(1) disulfides in ligand binding is supported by the effect of dithiothreitol (DTT). DTT breaks disulfide bonds, thereby strongly inhibiting ligand binding in AT(1) receptors. Despite retaining the same cysteines, AT(2) receptor ligand binding is paradoxically enhanced by DTT. Thus, we constructed a series of AT(2) cysteine mutations, either individually or paired, to establish the role of the cysteines and the source of DTT's effects. The AT(2) cysteine mutants surprisingly confirmed that the cysteines form disulfide bonds in the same manner as in the AT(1) subtype. However, breaking the AT(2) disulfide bridges yielded two responses. As in AT(1) receptors, mutations disrupting the disulfide bond between the first and second extracellular loops reduced AT(2) binding by 4-fold. In contrast, mutations breaking the disulfide bridge between the amino terminus and the third extracellular loop increased AT(2) binding, mimicking DTT's effect on this subtype. Further analysis of AT(1)/AT(2) chimeric exchange mutants of these domains suggested that the AT(2) amino terminus and third extracellular loop may possess latent binding epitopes that are only uncovered after DTT exposure.     

Importance of cysteines in the LDLR-related domain of the subgroup A avian leukosis and sarcoma virus receptor for viral entry.

The extracellular domain of the subgroup A avian sarcoma and leukemia virus (ALSV-A) receptor contains a region that is related in sequence to the ligand-binding motifs of the low-density lipoprotein receptor (LDLR). This domain contains six cysteines that are highly conserved between different members of the LDLR protein superfamily, and these residues are presumed to participate in intrachain disulfide bonds. To assess the importance of each cysteine in the ALSV-A receptor, individual or multiple cysteines were mutated to alanines and the altered receptors were tested for the ability to confer susceptibility to viral infection. Receptors bearing single mutations allowed subgroup A viral entry, albeit at less than wild-type levels. Receptors containing two or three substitutions were completely inactive if one of the changed residues was Cys-35 or Cys-50. Of the altered receptors tested, the only exception to this rule was a functional receptor which lacked both Cys-35 and Cys-50, an activity that was dependent on the presence of other cysteines in this protein. Most interestingly, a receptor containing both Cys-35 and Cys-50 but lacking the other four cysteines was completely functional. These results demonstrate the importance of Cys-35 and Cys-50 for viral entry mediated by the ALSV-A receptor and show that in the presence of these two residues, all of the other cysteines in this protein can be removed without loss of this function.     

Mutational analysis of disulfide bridges in the Mr 46,000 mannose 6-phosphate receptor. Localization and role for ligand binding

Formation of intramolecular disulfide bonds is a key step in the early maturation of newly synthesized Mr 46,000 mannose 6-phosphate receptors to acquire ligand-binding activity (Hille, A., Waheed, A., and von Figura, K. (1990) J. Cell Biol. 110, 963-972). The luminal domain of the receptor, which carries the ligand-binding site, contains 6 cysteine residues. We have analyzed the function of individual cysteine residues for the ligand-binding conformation by exchanging cysteine for glycine. In each case, the replacement of cysteine resulted in a complete loss of binding activity, indicating that all 6 luminal cysteine residues are required for the ligand-binding conformation. The cysteine mutants displayed a greatly reduced immunoreactivity, decreased stability, and a blocked or delayed transport to the trans Golgi. The glycosylation pattern allowed the distinguishing of three phenotypes, each of which was represented by one pair of cysteine mutants. Based on the assumption that replacement of either of the 2 cysteine residues forming a disulfide bond results in an identical phenotype, we postulate that disulfide bonds are formed between Cys-32 and Cys-78 and between Cys-132 and Cys-167, as well as between Cys-145 and Cys-179. This assumption was supported by the observation that the simultaneous exchange of the 2 cysteine residues of a putative pair resulted in the same phenotypes as the single exchange of either of the 2 cysteine residues.     

Conformational changes that occur during M3 muscarinic acetylcholine receptor activation probed by the use of an in situ disulfide cross-linking strategy.

The structural changes involved in ligand-dependent activation of G protein-coupled receptors are not well understood at present. To address this issue, we developed an in situ disulfide cross-linking strategy using the rat M(3) muscarinic receptor, a prototypical G(q)-coupled receptor, as a model system. It is known that a tyrosine residue (Tyr(254)) located at the C terminus of transmembrane domain (TM) V and several primarily hydrophobic amino acids present within the cytoplasmic portion of TM VI play key roles in determining the G protein coupling selectivity of the M(3) receptor subtype. To examine whether M3 receptor activation involves changes in the relative orientations of these functionally critical residues, pairs of cysteine residues were substituted into a modified version of the M(3) receptor that contained a factor Xa cleavage site within the third intracellular loop and lacked most endogenous cysteine residues. All analyzed mutant receptors contained a Y254C point mutation and a second cysteine substitution within the segment Lys(484)-Ser(493) at the intracellular end of TM VI. Following their transient expression in COS-7 cells, mutant receptors present in their native membrane environment (in situ) were subjected to mild oxidizing conditions, either in the absence or in the presence of the muscarinic agonist, carbachol. The successful formation of disulfide cross-links was monitored by studying changes in the electrophoretic mobility of oxidized, factor Xa-treated receptors on SDS gels. The observed cross-linking patterns indicated that M(3) receptor activation leads to structural changes that allow the cytoplasmic ends of TM V and TM VI to move closer to each other and that also appear to involve a major change in secondary structure at the cytoplasmic end of TM VI. This is the first study employing an in situ disulfide cross-linking strategy to examine agonist-dependent dynamic structural changes in a G protein-coupled receptor.     

Identification of an agonist-induced conformational change occurring adjacent to the ligand-binding pocket of the M(3) muscarinic acetylcholine receptor

To study the conformational changes that convert G protein-coupled receptors (GPCRs) from their resting to their active state, we used the M(3) muscarinic acetylcholine receptor, a prototypical class A GPCR, as a model system. Specifically, we employed a recently developed in situ disulfide cross-linking strategy that allows the formation of disulfide bonds in Cys-substituted mutant M(3) muscarinic receptors present in their native membrane environment. At present, little is known about the conformational changes that GPCR ligands induce in the immediate vicinity of the ligand-binding pocket. To address this issue, we generated 11 Cys-substituted mutant M(3) muscarinic receptors and characterized these receptors in transfected COS-7 cells. All analyzed mutant receptors contained an endogenous Cys residue (Cys-532(7.42)) located within the exofacial segment of transmembrane domain (TM) VII, close to the agonist-binding site. In addition, all mutant receptors harbored a second Cys residue that was introduced into the exofacial segment of TM III, within the sequence Leu-142(3.27)-Asn-152(3.37). Disulfide cross-linking studies showed that muscarinic agonists, but not antagonists, promoted the formation of a disulfide bond between S151(3.36)C and Cys-532. A three-dimensional model of the inactive state of the M(3) muscarinic receptor indicated that Cys-532 and Ser-151 face each other in the center of the TM receptor core. Our cross-linking data therefore support the concept that agonist activation pulls the exofacial segments of TMs VII and III closer to each other. This structural change may represent one of the early conformational events triggering the more pronounced structural reorganization of the intracellular receptor surface. To the best of our knowledge, this is the first direct demonstration of a conformational change occurring in the immediate vicinity of the binding site of a GPCR activated by a diffusible ligand.     

Mutationally induced disulfide bond formation within the third extracellular loop causes melanocortin 4 receptor inactivation in patients with obesity

By screening patients with severe early onset obesity for mutations within the melanocortin 4 receptor (MC4R) gene, we have identified a missense mutation (C271R) that occurs homozygous in two siblings with obesity. In-depth functional characterization of C271R revealed a right-shifted concentration response curve due to lower affinity to natural and synthetic MC4R agonists and a reduced cell surface expression. Cys-271 is located in the third extracellular loop. Here, we provide evidence that Cys-271 forms an intra-loop disulfide bond with Cys-277. Unexpectedly, we found that loss of receptor function is not only caused by the disruption of this disulfide bridge. Our data strongly support a new mechanism in which the receptor malfunction in the C271R mutant is induced by formation of a functionally disastrous disulfide bridge between Cys-277 and a third Cys residue at position 279. Mutational and chemical disruption of this improper disulfide bond was able to restore normal receptor potency. By demonstrating that a loss of a disulfide bond-participating Cys residue can favor a functionally disastrous disulfide bond, we now add a new mechanism of how Cys residues can be involved in G-protein-coupled receptor malfunction.     

Location of intrachain disulfide bonds in the VP5* and VP8* trypsin cleavage fragments of the rhesus rotavirus spike protein VP4.

Because the rotavirus spike protein VP4 contains conserved Cys residues at positions 216, 318, 380, and 774 and, for many animal rotaviruses, also at position 203, we sought to determine whether disulfide bonds were structural elements of VP4. Electrophoretic analysis of untreated and trypsin-treated rhesus rotavirus (RRV) and simain rotavirus SA11 in the presence and absence of the reducing agent dithioerythritol revealed that VP4 and its cleavage fragments VP5* and VP8* possessed intrachain disulfide bonds. Given that the VP8* fragments of RRV and SA11 contain only two Cys residues, those at positions 203 and 216, these data indicated that these two residues were covalently linked. Electrophoretic examination of truncated species of VP4 and VP4 containing Cys-->Ser mutations synthesized in reticulocyte lysates provided additional evidence that Cys-203 and Cys-216 in VP8* of RRV were linked by a disulfide bridge. VP5* expressed in vitro was able to form a disulfide bond analogous to that in the VP5* fragment of trypsin-treated RRV. Analysis of a Cys-774-->Ser mutant of VP5* showed that, while it was able to form a disulfide bond, a Cys-318-->Ser mutant of VP5* was not. These results indicated that the VP4 component of all rotaviruses, except B223, contains a disulfide bond that links Cys-318 and Cys-380 in the VP5* region of the protein. This bond is located between the trypsin cleavage site and the putative fusion domain of VP4. Because human rotaviruses lack Cys-203 and, hence, unlike many animal rotaviruses cannot possess a disulfide bond in VP8*, it is apparent that VP4 is structurally variable in nature, with human rotaviruses generally containing one disulfide linkage and animal rotaviruses generally containing two such linkages. Considered with the results of anti-VP4 antibody mapping studies, the data suggest that the disulfide bond in VP5* exists within the 2G4 epitope and may be located at the distal end of the VP4 spike on rotavirus particles.     

Role of extracellular cysteine residues in the adenosine A<Subscript>2A</Subscript> receptor

The G protein-coupled A_2A adenosine receptor represents an important drug target. Crystal structures and modeling studies indicated that three disulfide bonds are formed between ECL1 and ECL2 (I, Cys71^2.69-Cys159^45.43; II, Cys74^3.22-Cys146^45.30, and III, Cys77^3.25-Cys166^45.50). However, the A_2BAR subtype appears to require only disulfide bond III for proper function. In this study, each of the three disulfide bonds in the A_2AAR was disrupted by mutation of one of the cysteine residues to serine. The mutant receptors were stably expressed in Chinese hamster ovary cells and analyzed in cyclic adenosine monophosphate (cAMP) accumulation and radioligand binding studies using structurally diverse agonists: adenosine, NECA, CGS21680, and PSB-15826. Results were rationalized by molecular modeling. The observed effects were dependent on the investigated agonist. Loss of disulfide bond I led to a widening of the orthosteric binding pocket resulting in a strong reduction in the potency of adenosine, but not of NECA or 2-substituted nucleosides. Disruption of disulfide bond II led to a significant reduction in the agonists’ efficacy indicating its importance for receptor activation. Disulfide bond III disruption reduced potency and affinity of the small adenosine agonists and NECA, but not of the larger 2-substituted agonists. While all the three disulfide bonds were essential for high potency or efficacy of adenosine, structural modification of the nucleoside could rescue affinity or efficacy at the mutant receptors. At present, it cannot be excluded that formation of the extracellular disulfide bonds in the A_2AAR is dynamic. This might add another level of G protein-coupled receptor (GPCR) modulation, in particular for the cysteine-rich A_2A and A_2BARs.     

Versatility and differential roles of cysteine residues in human prostacyclin receptor structure and function

Prostacyclin plays important roles in vascular homeostasis, promoting vasodilatation and inhibiting platelet thrombus formation. Previous studies have shown that three of six cytoplasmic cysteines, particularly those within the C-terminal tail, serve as important lipidation sites and are differentially conjugated to palmitoyl and isoprenyl groups (Miggin, S. M., Lawler, O. A., and Kinsella, B. T. (2003) J. Biol. Chem. 278, 6947-6958). Here we report distinctive roles for extracellular- and transmembrane-located cysteine residues in human prostacyclin receptor structure-function. Within the extracellular domain, all cysteines (4 of 4) appear to be involved in disulfide bonding interactions (i.e. a highly conserved Cys-92-Cys-170 bond and a putative non-conserved Cys-5-Cys-165 bond), and within the transmembrane (TM) region there are several cysteines (3 of 8) that maintain critical hydrogen bonding interactions (Cys-118 (TMIII), Cys-251 (TMVI), and Cys-202 (TMV)). This study highlights the necessity of sulfhydryl (SH) groups in maintaining the structural integrity of the human prostacyclin receptor, as 7 of 12 extracellular and transmembrane cysteines studied were found to be differentially indispensable for receptor binding, activation, and/or trafficking. Moreover, these results also demonstrate the versatility and reactivity of these cysteine residues within different receptor environments, that is, extracellular (disulfide bonds), transmembrane (H-bonds), and cytoplasmic (lipid conjugation).     

The crystal structure of TrxA(CACA): Insights into the formation of a [2Fe-2S] iron-sulfur cluster in an Escherichia coli thioredoxin mutant

Escherichia coli thioredoxin is a small monomeric protein that reduces disulfide bonds in cytoplasmic proteins. Two cysteine residues present in a conserved CGPC motif are essential for this activity. Recently, we identified mutations of this motif that changed thioredoxin into a homodimer bridged by a [2Fe-2S] iron-sulfur cluster. When exported to the periplasm, these thioredoxin mutants could restore disulfide bond formation in strains lacking the entire periplasmic oxidative pathway. Essential for the assembly of the iron-sulfur was an additional cysteine that replaced the proline at position three of the CGPC motif. We solved the crystalline structure at 2.3 Angstroms for one of these variants, TrxA(CACA). The mutant protein crystallized as a dimer in which the iron-sulfur cluster is replaced by two intermolecular disulfide bonds. The catalytic site, which forms the dimer interface, crystallized in two different conformations. In one of them, the replacement of the CGPC motif by CACA has a dramatic effect on the structure and causes the unraveling of an extended alpha-helix. In both conformations, the second cysteine residue of the CACA motif is surface-exposed, which contrasts with wildtype thioredoxin where the second cysteine of the CXXC motif is buried. This exposure of a pair of vicinal cysteine residues apparently allows thioredoxin to acquire an iron-sulfur cofactor at its active site, and thus a new activity and mechanism of action.     

Structural insights into the role of the ACTH receptor cysteine residues on receptor function

The ACTH receptor, also known as the melanocortin-2 receptor (MC2R), is critical for ACTH-mediated adrenal glucocorticoid release. Human MC2R (hMC2R) has 10 cysteine residues, which are located in extracellular loops (ELs), transmembrane domains (TMs), and intracellular loops (ILs). In this study, we examined the importance of these cysteine residues in receptor function and determined their involvement in disulfide bond formation. We replaced these cysteines with serine and expressed the mutated receptors in adrenal OS3 cells, which lack endogenous MC2R. Our results indicate that four mutations, C21S in NH(2) terminus, C245S, C251S, and C253S in EL3, resulted in significant decrease both in receptor expression and receptor function. Mutation of cysteine 231 in TM6 significantly decreased ACTH binding affinity and potency. In contrast, the five other mutated receptors (C64S, C158S, C191S, C267S, and C293S) did not significantly alter ACTH binding affinity and potency. These results suggest that extracellular cysteine residue 21, 245, 251, and 253, as well as transmembrane cysteine residue 231 are crucial for ACTH binding and signaling. Further experiments suggest that a disulfide bond exists between the residue C245 and C251 in EL3. These findings provide important insights into the importance of cysteine residues of hMC2R for receptor function.     

Mutational analysis of predicted extracellular domains of human growth hormone secretagogue receptor 1a

The Class A family of guanine nucleotide-binding protein (G protein)-coupled receptors that includes receptors for motilin, ghrelin, and growth hormone secretagogue (GHS) has substantial potential importance as drug targets. Understanding of the molecular basis of hormone binding and receptor activation should provide insights helpful in the development of such drugs. We previously reported that Cys residues and the perimembranous residues in the extracellular loops and amino-terminal tail of the motilin receptor are critical for peptide ligand, motilin, binding and biological activity. In the current work, we focused on the predicted extracellular domains of the human GHS receptor 1a, and identified functionally important residues by using sequential deletions ranging from one to twelve amino acid residues and site-directed replacement mutagenesis approach. Each construct was transiently expressed in COS cells, and characterized for ghrelin- and growth hormone releasing peptide (GHRP)-6-stimulated intracellular calcium responses and ghrelin radioligand binding. Cys residues in positions 116 and 198 in the first and second extracellular loops and the perimembranous Glu¹⁸⁷ residue in the second extracellular loop were critical for ghrelin and GHRP-6 biological activity. These results suggest that Cys residues in the extracellular domains in this family of Class A G protein-coupled receptor is likely involved in the highly conserved and functionally important disulfide bond, and that the perimembranous residues contribute peptide ligand binding and signaling.