Characterization of the molecular mechanisms of the coupling between intracellular loops of prostacyclin receptor with the C-terminal domain of the Galphas protein in human coronary artery smooth muscle cells

The C-terminal domain of the Gs protein alpha subunit (Galphas Ct) and the first intracellular loop (iLP1) of prostacyclin receptor (IP) have been predicted to be involved in the receptor signaling mediated through the IP/Gs protein coupling by our previous NMR studies using synthetic peptides. To test whether the results of the peptide studies can be applied to the protein interaction between the IP receptor and the Gs protein in cells, a minigene technique was used to construct cDNAs that encoded either the amino acid residues of the Galphas or that of the individual intracellular loops of the IP receptor. The effects of the minigene-expressed protein fragments on cAMP production mediated by the IP/Gs coupling were evaluated through experiments that co-expressed peptides either through the Galphas Ct or the IP intracellular loops with the IP receptor in HEK293 cells. The first (iLP1) and third (iLP3) IP intracellular loops, as well as the Galphas Ct, which are important to the IP/Gs coupling-mediated signaling, were identified by the significant reduction of cAMP production when the corresponding peptides were expressed in the cells. Furthermore, the cAMP productions were significantly impaired in Galphas-knockout cells co-expressing the IP receptor with the Galphas C-terminal mutants (E392A, L393A and L394A), compared with the Galphas wild type. Blocking of the endogenous IP/Gs coupling by the minigene-expressed peptides of the Galphas CT, iLP1 and iLP3 was further observed in the human coronary artery smooth muscle cells (SMCs). These results indicate that the three residues (E392-L394) of the Galphas protein predicted from NMR peptide studies, and the IP iLP1 and iLP3 play important roles in the Galphas-mediated IP receptor signaling in the cells, which may be a general binding site for the corresponding regions of the other prostanoid receptors that couple to Gs protein.     

Function of the cytoplasmic tail of human calcitonin receptor-like receptor in complex with receptor activity-modifying protein 2

Receptor activity-modifying protein 2 (RAMP2) enables calcitonin receptor-like receptor (CRLR) to form an adrenomedullin (AM)-specific receptor. Here we investigated the function of the cytoplasmic C-terminal tail (C-tail) of human (h)CRLR by co-transfecting its C-terminal mutants into HEK-293 cells stably expressing hRAMP2. Deleting the C-tail from CRLR disrupted AM-evoked cAMP production or receptor internalization, but did not affect [¹²⁵I]AM binding. We found that CRLR residues 428-439 are required for AM-evoked cAMP production, though deleting this region had little effect on receptor internalization. Moreover, pretreatment with pertussis toxin (100 ng/mL) led to significant increases in AM-induced cAMP production via wild-type CRLR/RAMP2 complexes. This effect was canceled by deleting CRLR residues 454-457, suggesting Gi couples to this region. Flow cytometric analysis revealed that CRLR truncation mutants lacking residues in the Ser/Thr-rich region extending from Ser⁴⁴⁹ to Ser⁴⁶⁷ were unable to undergo AM-induced receptor internalization and, in contrast to the effect on wild-type CRLR, overexpression of GPCR kinases-2, -3 and -4 failed to promote internalization of CRLR mutants lacking residues 449-467. Thus, the hCRLR C-tail is crucial for AM-evoked cAMP production and internalization of the CRLR/RAMP2, while the receptor internalization is dependent on the aforementioned GPCR kinases, but not Gs coupling.     

Novel calcitonin-(8-32)-sensitive adrenomedullin receptors derived from co-expression of calcitonin receptor with receptor activity-modifying proteins

We tested whether heterodimers comprised of calcitonin (CT) receptor lacking the 16-amino acid insert in intracellular domain 1 (CTR(I1-)) and receptor activity-modifying protein (RAMP) can function not only as calcitonin gene-related peptide (CGRP) receptors but also as adrenomedullin (AM) receptors. Whether transfected alone or together with RAMP, human (h)CTR(I1-) appeared mainly at the surface of HEK-293 cells. Expression of CTR(I1-) alone led to significant increases in cAMP in response to hCGRP or hAM, though both peptides remained about 100-fold less potent than hCT. However, the apparent potency of AM, like that of CGRP, approached that of CT when CTR(I1-) was co-expressed with RAMP. CGRP- or AM-evoked cAMP production was strongly inhibited by salmon CT-(8-32), a selective amylin receptor antagonist, but not by hCGRP-(8-37) or hAM-(22-52), antagonists of CGRP and AM receptors, respectively. Moreover, the inhibitory effects of CT-(8-32) were much stronger in cells co-expressing CTR(I1-) and RAMP than in cells expressing CTR(I1-) alone. Co-expression of CTR(I1-) with RAMP thus appears to produce functional CT-(8-32)-sensitive AM receptors.     

Regulation of Constitutive GPR3 Signaling and Surface Localization by GRK2 and β-arrestin-2 Overexpression in HEK293 Cells

G protein-coupled receptor 3 (GPR3) is a constitutively active receptor that maintains high 3′-5′-cyclic adenosine monophosphate (cAMP) levels required for meiotic arrest in oocytes and CNS function. Ligand-activated G protein-coupled receptors (GPCRs) signal at the cell surface and are silenced by phosphorylation and β-arrestin recruitment upon endocytosis. Some GPCRs can also signal from endosomes following internalization. Little is known about the localization, signaling, and regulation of constitutively active GPCRs. We demonstrate herein that exogenously-expressed GPR3 localizes to the cell membrane and undergoes internalization in HEK293 cells. Inhibition of endocytosis increased cell surface-localized GPR3 and cAMP levels while overexpression of GPCR-Kinase 2 (GRK2) and β-arrestin-2 decreased cell surface-localized GPR3 and cAMP levels. GRK2 by itself is sufficient to decrease cAMP production but both GRK2 and β-arrestin-2 are required to decrease cell surface GPR3. GRK2 regulates GPR3 independently of its kinase activity since a kinase inactive GRK2-K220R mutant significantly decreased cAMP levels. However, GRK2-K220R and β-arrestin-2 do not diminish cell surface GPR3, suggesting that phosphorylation is required to induce GPR3 internalization. To understand which residues are targeted for desensitization, we mutated potential phosphorylation sites in the third intracellular loop and C-terminus and examined the effect on cAMP and receptor surface localization. Mutation of residues in the third intracellular loop dramatically increased cAMP levels whereas mutation of residues in the C-terminus produced cAMP levels comparable to GPR3 wild type. Interestingly, both mutations significantly reduced cell surface expression of GPR3. These results demonstrate that GPR3 signals at the plasma membrane and can be silenced by GRK2/β-arrestin overexpression. These results also strongly implicate the serine and/or threonine residues in the third intracellular loop in the regulation of GPR3 activity.     

Differential determinants for coupling of distinct G proteins with the class B secretin receptor

The secretin receptor is a prototypic class B G protein-coupled receptor that is activated by binding of its natural peptide ligand. The signaling effects of this receptor are mediated by coupling with Gs, which activates cAMP production, and Gq, which activates intracellular calcium mobilization. We have explored the molecular basis for the coupling of each of these G proteins to this receptor using systematic site-directed mutagenesis of key residues within each of the intracellular loop regions, and studying ligand binding and secretin-stimulated cAMP and calcium responses. Mutation of a conserved histidine in the first intracellular loop (H157A and H157R) markedly reduced cell surface expression, resulting in marked reduction in cAMP and elimination of measurable calcium responses. Mutation of an arginine (R153A) in the first intracellular loop reduced calcium, but not cAMP responses. Mutation of a dibasic motif in the second intracellular loop (R231A/K232A) had no significant effects on any measured responses. Mutations in the third intracellular loop involving adjacent lysine and leucine residues (K302A/L303A) or two arginine residues separated by a leucine and an alanine (R318A/R321A) significantly reduced cAMP responses, while the latter also reduced calcium responses. Additive effects were elicited by combining the effective mutations, while combining all the effective mutations resulted in a construct that continued to bind secretin normally, but that elicited no significant cAMP or calcium responses. These data suggest that, while some receptor determinants are clearly shared, there are also distinct determinants for coupling with each of these G proteins.     

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.     

Effects of reciprocal chimeras between the C-terminal portion of third intracellular loops of the human dopamine D2 and D3 receptors

The dopamine D3 receptor is a member of the G protein-coupled superfamily of receptors. However, its coupling with intracellular events is still not well understood. We have performed chimera constructions in which amino acid residues located in a region of the receptor involved in the coupling with second messengers (the C-terminal portion of the third intracellular loop) have been exchanged between dopamine D2 and D3 receptors. Chimera constructions did not modify substantially the pharmacological profiles, nor G protein coupling, as compared to their respective wild-type receptors. However, the D2 receptor chimera, containing the C-terminal portion of the third intracellular loop of the D3 receptor, has a lower potency to inhibit cyclic AMP production. The reciprocal construction generated a D3 receptor that is fully coupled to this second messenger pathway whereas, the native D3 receptor is uncoupled to this pathway in our transfected cells. These results suggest that the sequence selected is important for specific coupling characteristics shown by these two dopamine receptor homologues.     

Chimeric exchanges within the bradykinin B2 receptor intracellular face with the prostaglandin EP2 receptor as the donor: importance of the second intracellular loop for cAMP synthesis

The prostaglandin E2 (PGE(2)) EP2 receptor (EP2R) type is G protein coupled (GPCR) and links to Galphas. Through this receptor PGE(2) activates cAMP production. The bradykinin (BK) B2 receptor (BKB2R) is also a GPCR but links to Galphaq and Galphai and does not activate cAMP production in response to bradykinin. In an attempt to convert the BKB2R into a Galphas-linked adenylate cyclase-activating receptor we proceeded to make global and discrete motif replacements of the intracellular (IC) face of the BKB2R with the corresponding regions of the human EP2R. With this approach we produced hybrid receptors which, when stably transfected into wild type (WT) Rat-1 cells, bound BK but produced cAMP. Replacement of the second loop (IC2), third loop (IC3), the entire C terminus, and the distal C terminus resulted in receptors which bound BK. However, only the IC2 and IC3 exchanges resulted in cAMP-producing receptors. Of these two regions, the IC2 exchange was by far the better cAMP-generating receptor, producing cAMP at approximately 6.6-fold above WT BKB2R or approximately one fourth the amount produced by WT EP2R-transfected Rat-1 cells. Both human and rat EP2R and human beta2-adrenergic receptor exchanges of the IC2 produced equal quantities of cAMP. Focusing on the rBKB2R/hEP2R IC2 chimeras, the region consisting of residues 136-147 (BKB2R residue numbering) proved to contain a cAMP-generating motif. Within this region, the proximal six amino acids from the EP2R (HPYFYQ) at position 136-141 proved crucial for cAMP production (10-fold over WT BKB2R). The distal part of this region, the six residues at 142-147, played no role in cAMP production. On the other hand, the ALV motif of the BKB2R IC2, residues 133-135, proved important with respect to phosphatydilinositol (PI) turnover. Replacing the entire IC2 of BKB2R resulted in poor PI turnover, while including the AVL of BKB2R retained approximately half of the WT PI turnover. With respect to receptor uptake, all the IC2 mutants endocytosed as WT BKB2R (60% in 1h). However, the exchange of the distal and the whole C termini resulted in a marked drop in endocytosis (30% in 1h). These results demonstrate that the construction of a cAMP-producing BKB2/EP2 receptor hybrid is possible, with the IC2 region distal to DRYLALV proving important to Galphas linkage and the LALV motif within the IC2 of BKB2R and the region proximal to it proving important for Galphaq and Galphai linkage. Additionally, our results confirm the importance of the distal C terminus in determining receptor uptake.     

Post-activation-mediated changes in opioid receptors detected by N-terminal antibodies

The majority of studies examining activity-induced conformational changes in G protein-coupled receptors have focused on transmembrane helices or intracellular regions. Relatively few studies have examined the involvement of the extracellular region in general and the N-terminal region in particular in this process. To begin to address this, we generated a series of antibodies to the N-terminal region of opioid receptors. Characterization of these antibodies revealed that they differentially recognize activated receptors. Recently, we generated monoclonal antibodies that recognize regions proximal to glycosylation sites in the receptor N terminus. Characterization of these antibodies revealed that agonist treatment leads to a decrease in epitope recognition by the antibody presumably because of a movement of the region of the N terminus proximal to glycosylation sites. The time course of the decrease in antibody recognition suggested that it could be due to a post-activation-mediated event. Examination of the involvement of receptor residues in the C-tail and beta-arrestin binding using site-directed mutagenesis and cells or tissues lacking beta-arrestin 2 suggests a role for these desensitization-related mechanisms in governing antibody binding to the receptor. Thus, these N-terminally directed antibodies can differentially recognize post-activation-mediated changes in the C-terminal (intracellular) region of the receptor. Therefore, these conformation-sensitive antibodies represent powerful reagents to probe receptor activation states and provide a potential tool for identifying and characterizing new compounds of therapeutic interest.     

Two cytoplasmic loops of the glucagon receptor are required to elevate cAMP or intracellular calcium.

The glucagon receptor is a member of a distinct class of G protein-coupled receptors (GPCRs) sharing little amino acid sequence homology with the larger rhodopsin-like GPCR family. To identify the components of the glucagon receptor necessary for G-protein coupling, we replaced sequentially all or part of each intracellular loop (i1, i2, and i3) and the C-terminal tail of the glucagon receptor with the 11 amino acids comprising the first intracellular loop of the D4 dopamine receptor. When expressed in transiently transfected COS-1 cells, the mutant receptors fell into two different groups with respect to hormone-mediated signaling. The first group included the loop i1 mutants, which bound glucagon and signaled normally. The second group comprised the loop i2 and i3 chimeras, which caused no detectable adenylyl cyclase activation in COS-1 cells. However, when expressed in HEK 293T cells, the loop i2 or i3 chimeras caused very small glucagon-mediated increases in cAMP levels and intracellular calcium concentrations, with EC50 values nearly 100-fold higher than those measured for wild-type receptor. Replacement of both loops i2 and i3 simultaneously was required to completely abolish G protein signaling as measured by both cAMP accumulation and calcium flux assays. These results show that the i2 and i3 loops play a role in glucagon receptor signaling, consistent with recent models for the mechanism of activation of G proteins by rhodopsin-like GPCRs.     

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.     

Effects of truncations of the cytoplasmic tail of the luteinizing hormone/chorionic gonadotropin receptor on receptor-mediated hormone internalization.

The LH/CG receptor is a member of the family of G protein-coupled receptors and consists of a large N-terminal extracellular domain (which is responsible for binding hormone) attached to a region that spans the plasma membrane seven times, ending with an intracellularly located C-terminus. Binding of LH or human CG (hCG) to the LH/CG receptor causes a stimulation of adenylyl cyclase, presumably via activation of Gs. The binding of hormone also leads to its subsequent internalization by receptor-mediated endocytosis. In order to investigate the role of the cytoplasmic tail of this receptor in these events, we prepared a series of mutants in which progressively larger portions of the cytoplasmic tail were deleted. Deletion of 58 amino acids from the C-terminus, in which only 11 cytoplasmic residues remain, resulted in a receptor that was not expressed on the plasma membrane. Receptors rat LHR (rLHR)-t653 and rLHR-t631, in which 21 or 43 amino acids were removed, respectively, were properly expressed. These results suggest that a region(s) between residues 616 and 631 of the rLH/CG receptor are required for proper insertion and/or targeting of the receptor into the plasma membrane. Cells expressing rLHR-t653 or rLHR-t631 bound hCG with the same high affinity as cells expressing the full-length receptor, and basal levels of cAMP were the same among the cells. However, cells expressing the truncated receptors responded to hCG with approximately 2-fold greater levels of maximal cAMP accumulation than cells expressing the full-length receptor. Deletion of up to 43 amino acids from the C-terminus of the rLH/CG receptor had no deleterious effect on hCG internalization. In fact, mutants lacking 21 and 43 amino acids exhibited progressively faster rates of hCG internalization as compared to the full-length receptor. Once internalized, hCG was also degraded at a faster rate in cells expressing the truncated LH/CG receptors. Since hCG-stimulated cAMP stimulation and hCG internalization are retained by rLHR-t631, it can be concluded that the residues, not necessarily the same, required for these functions reside within the 26 amino acids of the cytoplasmic tail closest to the seventh transmembrane helix and/or residues within the intracellular loops. Our data show, however, that both hCG-stimulated cAMP production and hCG internalization are enhanced by the removal of the distal portion of the cytoplasmic tail.     

Functional analysis of the cytoplasmic domains of the human thyrotropin receptor by site-directed mutagenesis

The thyrotropin (TSH) receptor belongs to a family of guanine nucleotide protein-coupled receptors with seven transmembrane-spanning regions joined regulatory together by extracellular and intracellular loops. The cytoplasmic domain comprises three cytoplasmic loops and a cytoplasmic tail that are likely to be important in coupling of the receptor to the guanine nucleotide proteins. To address the question of which portions of the cytoplasmic domain of the TSH receptor are important in this process, we have altered groups of amino acids in the region of the TSH receptor by site-directed mutagenesis. Because of the low affinity of TSH binding to the TSH receptor mutated in the amino terminus of the second cytoplasmic loop and the amino terminus of the cytoplasmic tail, definitive conclusions cannot be made regarding the roles of these regions in signal transduction. However, our data indicate that the first cytoplasmic loop (residues 441-450), the carboxyl-terminal region of the second cytoplasmic loop (residues 528-537), and the carboxyl-terminal (but not the amino-terminal) region of the third cytoplasmic loop (residues 617-625) are important in the ability of the TSH receptor to mediate an increase in intracellular cAMP production. Furthermore, two-thirds of the carboxyl-terminal end of the cytoplasmic tail (residues 709-764; corresponding to the region not conserved between the TSH and lutropin/chorionic gonadotropin receptors) can be removed without functional impairment of the TSH receptor.     

The third intracellular loop of alpha 2-adrenergic receptors determines subtype specificity of arrestin interaction

Nonvisual arrestins (arrestin-2 and -3) serve as adaptors to link agonist-activated G protein-coupled receptors to the endocytic machinery. Although many G protein-coupled receptors bind arrestins, the molecular determinants involved in binding remain largely unknown. Because arrestins selectively promote the internalization of the alpha(2b)- and alpha(2c)-adrenergic receptors (ARs) while having no effect on the alpha(2a)AR, here we used alpha(2)ARs to identify molecular determinants involved in arrestin binding. Initially, we assessed the ability of purified arrestins to bind glutathione S-transferase fusions containing the third intracellular loops of the alpha(2a)AR, alpha(2b)AR, or alpha(2c)AR. These studies revealed that arrestin-3 directly binds to the alpha(2b)AR and alpha(2c)AR but not the alpha(2a)AR, whereas arrestin-2 only binds to the alpha(2b)AR. Truncation mutagenesis of the alpha(2b)AR identified two arrestin-3 binding domains in the third intracellular loop, one at the N-terminal end (residues 194-214) and the other at the C-terminal end (residues 344-368). Site-directed mutagenesis further revealed a critical role for several basic residues in arrestin-3 binding to the alpha(2b)AR third intracellular loop. Mutation of these residues in the holo-alpha(2b)AR and subsequent expression in HEK 293 cells revealed that the mutations had no effect on the ability of the receptor to activate ERK1/2. However, agonist-promoted internalization of the mutant alpha(2b)AR was significantly attenuated as compared with wild type receptor. These results demonstrate that arrestin-3 binds to two discrete regions within the alpha(2b)AR third intracellular loop and that disruption of arrestin binding selectively abrogates agonist-promoted receptor internalization.     

Structure/function analysis of alpha2A-adrenergic receptor interaction with G protein-coupled receptor kinase 2

G protein-coupled receptors (GPCRs) mediate the ability of a diverse array of extracellular stimuli to control intracellular signaling. Many GPCRs are phosphorylated by G protein-coupled receptor kinases (GRKs), a process that mediates agonist-specific desensitization in many cells. Although GRK binding to activated GPCRs results in kinase activation and receptor phosphorylation, relatively little is known about the mechanism of GRK/GPCR interaction or how this interaction results in kinase activation. Here, we used the alpha2A-adrenergic receptor (alpha(2A)AR) as a model to study GRK/receptor interaction because GRK2 phosphorylation of four adjacent serines within the large third intracellular loop of this receptor is known to mediate desensitization. Various domains of the alpha(2A)AR were expressed as glutathione S-transferase fusion proteins and tested for the ability to bind purified GRK2. The second and third intracellular loops of the alpha(2A)AR directly interacted with GRK2, whereas the first intracellular loop and C-terminal domain did not. Truncation mutagenesis identified three discrete regions within the third loop that contributed to GRK2 binding, the membrane proximal N- and C-terminal regions as well as a central region adjacent to the phosphorylation sites. Site-directed mutagenesis revealed a critical role for specific basic residues within these regions in mediating GRK2 interaction with the alpha(2A)AR. Mutation of these residues within the holo-alpha(2A)AR diminished GRK2-promoted phosphorylation of the receptor as well as the ability of the kinase to be activated by receptor binding. These studies provide new insight into the mechanism of interaction and activation of GRK2 by GPCRs and suggest that GRK2 binding is critical not only for receptor phosphorylation but also for full activity of the kinase.     

A comprehensive structure-function map of the intracellular surface of the human C5a receptor. I. Identification of critical residues

G protein-coupled receptors are one of the largest protein families in nature; however, the mechanisms by which they activate G proteins are still poorly understood. To identify residues on the intracellular face of the human C5a receptor that are involved in G protein activation, we performed a genetic analysis of each of the three intracellular loops and the carboxyl-terminal tail of the receptor. Amino acid substitutions were randomly incorporated into each loop, and functional receptors were identified in yeast. The third intracellular loop contains the largest number of preserved residues (positions resistant to amino acid substitutions), followed by the second loop, the first loop, and lastly the carboxyl terminus. Surprisingly, complete removal of the carboxyl-terminal tail did not impair C5a receptor signaling. When mapped onto a three-dimensional structural model of the inactive state of the C5a receptor, the preserved residues reside on one half of the intracellular surface of the receptor, creating a potential activation face. Together these data provide one of the most comprehensive functional maps of the intracellular surface of any G protein-coupled receptor to date.     

Alternate coupling of receptors to Gs and Gi in pancreatic and submandibular gland cells.

Many Gs-coupled receptors can activate both cAMP and Ca2+ signaling pathways. Three mechanisms for dual activation have been proposed. One is receptor coupling to both Gs and G15 (a Gq class heterotrimeric G protein) to initiate independent signaling cascades that elevate intracellular levels of cAMP and Ca+2, respectively. The other two mechanisms involve cAMP-dependent protein kinase-mediated activation of phospholipase Cbeta either directly or by switching receptor coupling from Gs to Gi. These mechanisms were primarily inferred from studies with transfected cell lines. In native cells we found that two Gs-coupled receptors (the vasoactive intestinal peptide and beta-adrenergic receptors) in pancreatic acinar and submandibular gland duct cells, respectively, evoke a Ca2+ signal by a mechanism involving both Gs and Gi. This inference was based on the inhibitory action of antibodies specific for Galphas, Galphai, and phosphatidylinositol 4,5-bisphosphate, pertussis toxin, RGS4, a fragment of beta-adrenergic receptor kinase and inhibitors of cAMP-dependent protein kinase. By contrast, Ca2+ signaling evoked by Gs-coupled receptor agonists was not blocked by Gq class-specific antibodies and was unaffected in Galpha15 -/- knockout mice. We conclude that sequential activation of Gs and Gi, mediated by cAMP-dependent protein kinase, may represent a general mechanism in native cells for dual stimulation of signaling pathways by Gs-coupled receptors.     

Dual intracellular pathways in gonadotropin releasing hormone (GNRH) induced desensitization of luteinizing hormone (LH) secretion.

The mechanisms of GnRH-induced desensitization of LH secretion are poorly understood. Protein kinase C (PKC) and protein kinase A (PKA) desensitize some receptors of the 7-membrane type, and the GnRH receptor has consensus phosphorylation sites for PKC in the first and third intracellular loops, and a site for PKA in the first intracellular loop. In the first set of experiments we determined whether synthetic peptides representing the three intracellular loops of the receptor could be phosphorylated in vitro by purified PKC and PKA. As compared with a model substrate peptide for PKC, the third intracellular loop was phosphorylated 74% and the first intracellular loop 21%; PKA-phosphorylated the first intracellular loop peptide 17% as well as a model peptide substrate. In the second set of experiments, we used phorbol 12-myristate 13 acetate (PMA), an established PKC stimulator, and cholera toxin (CTX), established to activate the Gs protein and presumed to activate PKA, to treat cultured rat pituitary cells followed by LH measurements. Treatment with both drugs severely impaired GnRH-stimulated LH secretion whereas neither drug alone reduced LH secretion. Dibutyryl cAMP did not duplicate the effects of cholera toxin suggesting that the CTX action could not be explained by an increase in cAMP. These results suggest that more than one intracellular signaling pathway requires activation in order to induce desensitization; one pathway involves PKC and the other involves a pathway stimulated by cholera toxin, presumably Gs protein, which does not involve PKA.