Role of the highly conserved Asp-Arg-Tyr motif in signal transduction of the CB2 cannabinoid receptor

The DRY motif, at the junction of transmembrane helix 3 and intracellular loop 2 of G protein-coupled receptors, is highly conserved. Mutations were introduced into the CB2 cannabinoid receptor to study the role of this motif in CB2 signaling. D mutations (DRY130-132AAA and D130A) markedly reduced binding of cannabinoid agonists, while no significant reduction was observed with R131A or Y132A. Mutating R (R131A) only partially reduced, and mutating Y (Y132A) more efficiently reduced the cannabinoid-induced inhibition of adenylyl cyclase. Thus, in CB2, D130 is involved in agonist binding, whereas Y seems to have a role in receptor downstream signaling.     

Functional role of tryptophan residues in the fourth transmembrane domain of the CB(2) cannabinoid receptor

Several tryptophan (Trp) residues are conserved in G protein-coupled receptors (GPCRs). Relatively little is known about the contribution of these residues and especially of those in the fourth transmembrane domain in the function of the CB(2) cannabinoid receptor. Replacing W158 (very highly conserved in GPCRs) and W172 (conserved in CB(1) and CB(2) cannabinoid receptors but not in many other GPCRs) of the human CB(2) receptor with A or L or with F or Y produced different results. We found that the conservative change of W172 to F or Y retained cannabinoid binding and downstream signaling (inhibition of adenylyl cyclase), whereas removal of the aromatic side chain by mutating W172 to A or L eliminated agonist binding. W158 was even more sensitive to being mutated. We found that the conservative W158F mutation retained wild-type binding and signaling activities. However, W158Y and W158A mutants completely lost ligand binding capacity. Thus, the Trp side chains at positions 158 and 172 seem to have a critical, but different, role in cannabinoid binding to the human CB(2) 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.     

Contribution of conserved glycine residues to ATP action at human P2X1 receptors: mutagenesis indicates that the glycine at position 250 is important for channel function

Glycine residues can introduce flexibility in proteins, give rise to turns and breaks in secondary structure and are key components of some nucleotide binding motifs. In the P2X receptor extracellular ATP binding domain, 11 glycine residues are completely conserved and an additional five are conserved in at least five of the seven family members. We have mutated individual conserved glycine residues and determined their effect on the ATP sensitivity and time-course of P2X1 receptors expressed in Xenopus oocytes. In the majority of cases, replacement by alanine had no or a less than 3-fold effect on ATP sensitivity and time-course of responses. G71A resulted in a 6-fold decrease in ATP potency and ATP (10 mM) failed to evoke functional responses from G96A, G250A and G301A mutant receptors. However, proline or cysteine could substitute for glycine at positions 96 and 301, giving receptors that were essentially normal. At glycine 250 substitution by serine gave functional responses to ATP with no effect on ATP sensitivity but a reduction in peak amplitude; in contrast, functional responses were not recorded when glycine 250 was replaced by the amino acids alanine, cysteine, aspartate, phenylalanine, isoleucine, lysine, proline or asparagine. These results suggest that glycine 250 plays an important role in determining the function of P2X receptors.     

Functional role of proline and tryptophan residues highly conserved among G protein-coupled receptors studied by mutational analysis of the m3 muscarinic receptor.

Most G protein-coupled receptors contain a series of highly conserved proline and tryptophan residues within their hydrophobic transmembrane domains (TMD I-VII). To study their potential role in ligand binding and receptor function, the rat m3 muscarinic acetylcholine receptor was used as a model system. A series of mutant receptors in which the conserved proline and tryptophan residues were individually replaced with alanine and phenylalanine, respectively, was created and transiently expressed in COS-7 cells. [3H]N-methylscopolamine ([3H]NMS) saturation binding studies showed that three of the seven mutant receptors studied (Pro242-->Ala, TMD V; Pro505-->Ala, TMD VI; Pro540-->Ala, TMD VII) were expressed at 35-100 times lower levels than the wild-type receptor while displaying 'm3-like' antagonist binding affinities. Pro201-->Ala (TMD IV) showed drastically reduced binding affinities (up to 450-fold) for both muscarinic agonists and antagonists. Whereas most mutant receptors retained strong functional activity, Pro540-->Ala (TMD VII) was found to be severely impaired in its ability to stimulate carbachol-induced phosphatidyl inositol hydrolysis (Emax approximately 25% of wild type m3). Interestingly, this mutant receptor bound muscarinic agonists with 7- to 19-fold higher affinities than the wild type receptor. The Trp-->Phe substitutions (Trp192-->Phe, TMD IV; Trp503-->Phe, TMD VI; Trp530-->Phe, TMD VII) resulted in less pronounced changes (compared with the Pro-->Ala mutant receptors) in both ligand binding and receptor function. Our data indicate that the proline residues that are highly conserved across the entire superfamily of G protein-coupled receptors play key roles in receptor expression, ligand binding and receptor activation.     

In human and rat lung membranes [35S]GTPgammaS binding is a tool for pharmacological characterization of G protein-coupled dinucleotide receptors.

The P2Y receptor family is activated by extracellular nucleotides such as ATP and UTP. P2Y receptors regulate physiological functions in numerous cell types. In lung, the P2Y2 receptor subtype plays a role in controlling Cl- and fluid transport. Besides ATP or UTP, also diadenosine tetraphosphate (Ap4A), a stable nucleotide, seems to be of physiological importance. In membrane preparations from human and rat lung we applied several diadenosine polyphosphates to investigate whether they act as agonists for G protein-coupled receptors. We assessed this by determining the stimulation of [35S]GTPgammaS binding. Stimulation of [35S]GTPgammaS binding to G proteins has already been successfully applied to elucidate agonist binding to various G protein-coupled receptors. Ap(n)A (n = 2 to 6) enhanced [35S]GTPgammaS binding similarly in human and rat lung membranes, an indication of the existence of G protein-coupled receptor binding sites specific for diadenosine polyphosphates. Moreover, in both human and rat lung membranes comparable pharmacological properties were found for a diadenosine polyphosphate ([3H]Ap4A) binding site. The affinity for Ap2A, Ap3A, Ap4A, Ap5A, and Ap6A was also comparable. 8-Diazido-Ap4A and ATP were less potent, whereas the pyrimidine nucleotide UTP showed hardly any affinity. Thus, we present evidence that different diadenosine polyphosphates bind to a common G protein-coupled receptor binding site in membranes derived either from human or rat lung.     

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.     

Mutagenesis studies of conserved proline residues of human P2X receptors for ATP indicate that proline 272 contributes to channel function

Proline residues can play a major role in the secondary structure of proteins. In the extracellular ATP binding loop of P2X receptors there are four totally conserved proline residues (P2X1 receptor numbering; P93, P166, P228 and P272) and three less conserved residues P196 (six of seven isoforms), P174 and P225 (five of seven isoforms). We have mutated individual conserved proline residues in the human P2X1 receptor and determined their properties. Mutants were expressed in Xenopus oocytes and characterized using a two-electrode voltage clamp. Mutants P166A, P174A, P196A, P225A and P228A had no effect on ATP potency compared with wild-type and P93A had a fourfold decrease in ATP potency. The P272A, P272D and P272K receptor mutants were expressed at the cell surface; however, these mutants were non-functional. In contrast, P272I, P272G and P272F produced functional channels, with either no effect or a 2.5- or 6.5-fold increase in ATP potency, respectively. At P272F receptors the apparent affinity of the ATP analogue antagonist 2',3'-O-(2,4,6-trinitrophenyl)-ATP was increased by 12.5-fold. These results suggest that individual proline residues are not essential for normal P2X receptor function and that the receptor conformation around P272 contributes to ATP binding at the receptor.     

Role of the conserved DRY motif on G protein activation of rat angiotensin II receptor type 1A

To delineate the functional importance of the highly conserved triplet amino acid sequence, Asp-Arg-Tyr (DRY) among G protein-coupled receptors in the second intracellular loop, these residues of rat angiotensin II (Ang II) receptor type 1A (AT(1A)) were changed by alanine or glycine by site-directed mutagenesis. These mutant receptors were stably expressed in CHO-K1 cells, and the binding of Ang II, GTP effect, InsP(3) production, and the acidification of the medium in response to Ang II were determined. The effects of GTPgammaS on Ang II binding in the mutant receptors D125A and D125G were markedly reduced. InsP(3) production of the mutant D125A, D125G, R126A, and R126G was markedly reduced. Extracellular acidification of D125A was not distinguishable from untransfected CHO-K1 cells. Mutant Y127A was able to produce InsP(3) and acidify medium comparable with wild type AT(1A). These results indicate as follows; Asp(125) is essential for intracellular signal transduction involving G protein coupling, Arg(126) is essential for coupling of G(q) protein but not other G proteins, and Tyr(127) is not important for G protein coupling.     

Congenital insulin resistance associated with a conformational alteration in a conserved beta-sheet in the insulin receptor L1 domain.

The hormone binding site of members of the insulin receptor family is contained within a highly conserved extracellular region of the receptor. Recent crystallization of the N-terminal region of the binding site revealed two large domains (L1, L2), each organized as a single-stranded right-handed beta-helix, connected by a rod-shaped cysteine-rich domain. Here, we analyze two new naturally occurring mutations in a single beta-sheet within L1, D59G and L62P, that we previously identified in a young woman with classic congenital insulin resistance (type A). Substitution of D59G, a beta-sheet connecting loop residue, caused decreased hormone binding but did not disrupt overall folding, assembly, or movement to the cell surface. In contrast, replacement of the adjacent residue L62P, which is located within the beta-sheet, and positioned in a hormone binding surface, completely disrupted intracellular folding, oligomerization, and trafficking and resulted in aberrant proteolytic degradation. Immunohistochemistry in combination with biosynthetic studies showed that misfolded receptors were retained in an incorrect cellular location and that they colocalized with the resident endoplasmic reticulum chaperone calnexin. This study, together with other mutagenesis data, shows that formation of beta-sheet elements within the L1 beta-helix are critical for the folding of the entire extracellular domain of the receptor and that the hormone contact site is composed in part by residues in this domain.     

A beta-arrestin binding determinant common to the second intracellular loops of rhodopsin family G protein-coupled receptors

beta-Arrestins have been shown to inhibit competitively G protein-dependent signaling and to mediate endocytosis for many of the hundreds of nonvisual rhodopsin family G protein-coupled receptors (GPCR). An open question of fundamental importance concerning the regulation of signal transduction of several hundred rhodopsin-like GPCRs is how these receptors of limited sequence homology, when considered in toto, can all recruit and activate the two highly conserved beta-arrestin proteins as part of their signaling/desensitization process. Although the serine and threonine residues that form GPCR kinase phosphorylation sites are common beta-arrestin-associated receptor determinants regulating receptor desensitization and internalization, the agonist-activated conformation of a GPCR probably reveals the most fundamental determinant mediating the GPCR and arrestin interaction. Here we identified a beta-arrestin binding determinant common to the rhodopsin family GPCRs formed from the proximal 10 residues of the second intracellular loop. We demonstrated by both gain and loss of function studies for the serotonin 2C, beta2-adrenergic, alpha2a)adrenergic, and neuropeptide Y type 2 receptors that the highly conserved amino acids, proline and alanine, naturally occurring in rhodopsin family receptors six residues distal to the highly conserved second loop DRY motif regulate beta-arrestin binding and beta-arrestin-mediated internalization. In particular, as demonstrated for the beta2 AR, this occurs independently of changes in GPCR kinase phosphorylation. These results suggest that a GPCR conformation directed by the second intracellular loop, likely using the loop itself as a binding patch, may function as a switch for transitioning beta-arrestin from its inactive form to its active receptor-binding state.     

Functional roles of conserved transmembrane prolines in the human VPAC(1) receptor

The importance of three conserved transmembrane prolines of the human vasoactive intestinal polypeptide (VPAC)(1) receptor was examined by single alanine substitution. P266A, P300A and P348A reduced the expression level, but maintained the binding to VIP. P266A showed decreased ability to stimulate cAMP, while P300A and P348A displayed an increased potency in cAMP production combined with a high sensitivity towards GTP compared to the wild type receptor. In addition, substitutions of two conserved leucines located in position -2 and +1 from P348 were investigated. L346A and L349A reduced the receptor expression, influenced the G protein coupling and decreased the receptor activity. These observations, which are the first on conserved transmembrane prolines within this family of receptors, indicate that these residues are important for receptor expression, G protein coupling and receptor activity.     

A membrane network of receptors and enzymes for adenine nucleotides and nucleosides

Most cells express more than one receptor plus degrading enzymes for adenine nucleotides or nucleosides, and cellular responses to purines are rarely compatible with the actions of single receptors. Therefore, these receptors are viewed as components of a combinatorial receptor web rather than self-dependent entities, but it remained unclear to what extent they can associate with each other to form signalling units. P2Y(1), P2Y(2), P2Y(12), P2Y(13), P2X(2), A(1), A(2A) receptors and NTPDase1 and -2 were expressed as fluorescent fusion proteins which were targeted to membranes and signalled like the unlabelled counterparts. When tested by FRET microscopy, all the G protein-coupled receptors proved able to form heterooligomers with each other, and P2Y(1), P2Y(12), P2Y(13), A(1), A(2A), and P2X(2) receptors also formed homooligomers. P2Y receptors did not associate with P2X, but G protein-coupled receptors formed heterooligomers with NTPDase1, but not NTPDase2. The specificity of prototypic interactions (P2Y(1)/P2Y(1), A(2A)/P2Y(1), A(2A)/P2Y(12)) was corroborated by FRET competition or co-immunoprecipitation. These results demonstrate that G protein-coupled purine receptors associate with each other and with NTPDase1 in a highly promiscuous manner. Thus, purinergic signalling is not only determined by the expression of receptors and enzymes but also by their direct interaction within a previously unrecognized multifarious membrane network.     

Receptor subtype-specific docking of Asp6.59 with C-terminal arginine residues in Y receptor ligands

Y receptors (YRs) are G protein-coupled receptors whose Y(1)R, Y(2)R, and Y(5)R subtypes preferentially bind neuropeptide Y (NPY) and peptide YY, whereas mammalian Y(4)Rs show a higher affinity for pancreatic polypeptide (PP). Comparison of YR orthologs and paralogs revealed Asp(6.59) to be fully conserved throughout all of the YRs reported so far. By replacing this conserved aspartic acid residue with alanine, asparagine, glutamate, and arginine, we now show that this residue plays a crucial role in binding and signal transduction of NPY/PP at all YRs. Sensitivity to distinct replacements is, however, receptor subtype-specific. Next, we performed a complementary mutagenesis approach to identify the contact site of the ligand. Surprisingly, this conserved residue interacts with two different ligand arginine residues by ionic interactions; although in Y(2)R and Y(5)R, Arg(33) is the binding partner of Asp(6.59), in Y(1)R and Y(4)R, Arg(35) of human PP and NPY interacts with Asp(6.59). Furthermore, Arg(25) of PP and NPY is involved in ligand binding only at Y(2)R and Y(5)R. This suggests significant differences in the docking of YR ligands between Y(1/4)R and Y(2/5)R and provides new insights into the molecular binding mode of peptide agonists at GPCRs. Furthermore, the proposed model of a subtype-specific binding mode is in agreement with the evolution of YRs.     

Functional screening of G protein-coupled receptors by measuring intracellular calcium with a fluorescence microplate reader

Ligand binding studies reveal information about affinity to G protein-coupled receptors (GPCRs) rather than functional properties. Increase in intracellular Ca(2+) appears to represent a universal second messenger signal for a majority of recombinant GPCRs. Here, we exploit Ca(2+) signaling as a fast and sensitive functional screening method for a number of GPCRs coupled to different G proteins. Ca(2+) fluorescence measurements are performed using Oregon Green 488 BAPTA-1/AM and a microplate reader equipped with an injector. Buffer alone or test compounds dissolved in buffer are injected into a cell suspension, and fluorescence intensity is recorded for 30 s. Each of the GPCRs tested--G(q)-coupled P2Y(2), G(s)-coupled dopamine D1 and D5, G(i)-coupled dopamine D2L, and G(q/11)-coupled muscarinic acetylcholine M1--yielded a significant rise in intracellular free [Ca(2+)] on agonist stimulation. Agonist stimulation was dose dependent, as shown for ATP or UTP stimulation of P2Y(2) receptors (EC(50) = 1 microM), SKF38393 stimulation of hD1 and hD5 (EC(50) = 18.1 nM and 2.7 nM), and quinpirole at hD2L (EC(50) = 6.5 nM). SCH23390 (at hD1 and hD5) and spiperone, haloperidol, and clozapine (at hD2L) competitively antagonized the Ca(2+) response. Furthermore, the Ca(2+) assay served to screen suramin analogs for antagonistic activity at P2Y(2) receptors. Screening at dopamine receptors revealed LE300, a new lead for a dopamine receptor antagonist. Advantages of the assay include fast and simple 96- or 384-well plate format (high-throughput screening), use of a visible light-excitable fluorescent dye, applicability to a majority of GPCRs, and simultaneous analysis of distinct Ca(2+) fluxes.     

Identification of the hydrophobic ligand binding pocket of the S1P1 receptor

Sphingosine 1-phosphate (S1P), a naturally occurring sphingolipid mediator and also a second messenger with growth factor-like actions in almost every cell type, is an endogenous ligand of five G protein-coupled receptors (GPCRs) in the endothelial differentiation gene family. The lack of GPCR crystal structures sets serious limitations to rational drug design and in silico searches for subtype-selective ligands. Here we report on the experimental validation of a computational model of the ligand binding pocket of the S1P1 GPCR surrounding the aliphatic portion of S1P. The extensive mutagenesis-based validation confirmed 18 residues lining the hydrophobic ligand binding pocket, which, combined with the previously validated three head group-interacting residues, now complete the mapping of the S1P ligand recognition site. We identified six mutants (L3.43G/L3.44G, L3.43E/L3.44E, L5.52A, F5.48G, V6.40L, and F6.44G) that maintained wild type [32P]S1P binding with abolished ligand-dependent activation by S1P. These data suggest a role for these amino acids in the conformational transition of S1P1 to its activated state. Three aromatic mutations (F5.48Y, F6.44G, and W6.48A) result in differential activation, by S1P or SEW2871, indicating that structural differences between the two agonists can partially compensate for differences in the amino acid side chain. The now validated ligand binding pocket provided us with a pharmacophore model, which was used for in silico screening of the NCI, National Institutes of Health, Developmental Therapeutics chemical library, leading to the identification of two novel nonlipid agonists of S1P1.     

Mutation of tyrosine in the conserved NPXXY sequence leads to constitutive phosphorylation and internalization, but not signaling, of the human B2 bradykinin receptor

Although the G protein-coupled receptors (GPCRs) share a similar seven-transmembrane domain structure, only a limited number of amino acid residues is conserved in their protein sequences. One of the most highly conserved sequences is the NPXXY motif located at the cytosolic end of the transmembrane region-7 of many GPCRs, particularly of those belonging to the family of the rhodopsin/beta-adrenergic-like receptors. Exchange of Tyr(305) in the corresponding NPLVY sequence of the bradykinin B(2) receptor (B(2)R) for Ala resulted in a mutant, termed Y305A, that internalized [(3)H]bradykinin (BK) almost as rapidly as the wild-type (wt) B(2)R. However, receptor sequestration of the mutant after stimulation with BK was clearly reduced relative to the wt B(2)R. Confocal fluorescence microscopy revealed that, in contrast to the B(2)R-enhanced green fluorescent protein chimera, the Y305A-enhanced green fluorescent protein chimera was predominantly located intracellularly even in the absence of BK. Two-dimensional phosphopeptide analysis showed that the mutant Y305A constitutively exhibited a phosphorylation pattern similar to that of the BK-stimulated wt B(2)R. Ligand-independent Y305A internalization was demonstrated by the uptake of rhodamine-labeled antibodies directed to a tag sequence at the N terminus of the mutant receptor. Co-immunoprecipitation revealed that Y305A is precoupled to G(q/11) without activating the G protein because the basal accumulation rate of inositol phosphate was unchanged as compared with wt B(2)R. We conclude, therefore, that the Y305A mutation of B(2)R induces a receptor conformation which is prone to ligand-independent phosphorylation and internalization. The mutated receptor binds to, but does not activate, its cognate heterotrimeric G protein G(q/11), thereby limiting the extent of ligand-independent receptor internalization.     

Functional and structural roles of conserved cysteine residues in the carboxyl-terminal domain of the follicle-stimulating hormone receptor in human embryonic kidney 293 cells

The carboxyl-terminal segment of G protein-coupled receptors has one or more conserved cysteine residues that are potential sites for palmitoylation. This posttranslational modification contributes to membrane association, internalization, and membrane targeting of proteins. In contrast to other members of the glycoprotein hormone receptor family (the LH and thyroid-stimulating hormone receptors), it is not known whether the follicle-stimulating hormone receptor (FSHR) is palmitoylated and what are the effects of abolishing its potential palmitoylation sites. In the present study, a functional analysis of the FSHR carboxyl-terminal segment cysteine residues was carried out. We constructed a series of mutant FSHRs by substituting cysteine residues with alanine, serine, or threonine individually and together at positions 629 and 655 (conserved cysteines) and 627 (nonconserved). The results showed that all three cysteine residues are palmitoylated but that only modification at Cys629 is functionally relevant. The lack of palmitoylation does not appear to greatly impair coupling to G(s) but, when absent at position 629, does significantly impair cell surface membrane expression of the partially palmitoylated receptor. All FSHR Cys mutants were capable of binding agonist with the same affinity as the wild-type receptor and internalizing on agonist stimulation. Molecular dynamics simulations at a time scale of approximately 100 nsec revealed that replacement of Cys629 resulted in structures that differed significantly from that of the wild-type receptor. Thus, deviations from wild-type conformation may potentially contribute to the severe impairment in plasma membrane expression and the modest effects on signaling exhibited by the receptors modified in this particular position.     

Autocatalytic cleavage of the EMR2 receptor occurs at a conserved G protein-coupled receptor proteolytic site motif

Post-translational cleavage at the G protein-coupled receptor proteolytic site (GPS) has been demonstrated in many class B2 G protein-coupled receptors as well as other cell surface proteins such as polycystin-1. However, the mechanism of the GPS proteolysis has never been elucidated. Here we have characterized the cleavage of the human EMR2 receptor and identified the molecular mechanism of the proteolytic process at the GPS. Proteolysis at the highly conserved His-Leu downward arrow Ser(518) cleavage site can occur inside the endoplasmic reticulum compartment, resulting in two protein subunits that associate noncovalently as a heterodimer. Site-directed mutagenesis of the P(+1) cleavage site (Ser(518)) shows an absolute requirement of a Ser, Thr, or Cys residue for efficient proteolysis. Substitution of the P(-2) His residue to other amino acids produces slow processing precursor proteins, which spontaneously hydrolyze in a defined cell-free system. Further biochemical characterization indicates that the GPS proteolysis is mediated by an autocatalytic intramolecular reaction similar to that employed by the N-terminal nucleophile hydrolases, which are known to activate themselves by self-catalyzed cis-proteolysis. We propose here that the autoproteolytic cleavage of EMR2 represents a paradigm for the other GPS motif-containing proteins and suggest that these GPS proteins belong to a cell surface receptor subfamily of N-terminal nucleophile hydrolases.     

Proper receptor signalling in a mutant catfish gonadotropin-releasing hormone receptor lacking the highly conserved Asp(90) residue

The negatively charged side chain of an Asp residue in transmembrane domain 2 is likely to play an important role in receptor signalling since it is highly conserved in the whole family of G protein-coupled receptors, except in mammalian gonadotropin-releasing hormone (GnRH) receptors. In this paper we show that the conserved Asp(90) of the catfish GnRH receptor can be substituted by a neutral Asn(90) without abolishing receptor signalling if another negatively charged Glu(93) is introduced in a proximal region of the receptor interior, thereby mimicking the Glu(90)-Lys(121) salt bridge of mammalian GnRH receptors.