Agonist-induced signaling, desensitization, and internalization of a phosphorylation-deficient AT1A angiotensin receptor

An analysis of the functional role of a diacidic motif (Asp236-Asp237) in the third intracellular loop of the AT1A angiotensin II (Ang II) receptor (AT1-R) revealed that substitution of both amino acids with alanine (DD-AA) or asparagine (DD-NN) residues diminished Ang II-induced receptor phosphorylation in COS-7 cells. However, Ang II-stimulated inositol phosphate production, mitogen-activated protein kinase, and AT1 receptor desensitization and internalization were not significantly impaired. Overexpression of dominant negative G protein-coupled receptor kinase 2 (GRK2)K220M decreased agonist-induced receptor phosphorylation by approximately 40%, but did not further reduce the impaired phosphorylation of DD-AA and DD-NN receptors. Inhibition of protein kinase C by bisindolylmaleimide reduced the phosphorylation of both the wild-type and the DD mutant receptors by approximately 30%. The inhibitory effects of GRK2K220M expression and protein kinase C inhibition by bisindolylmaleimide on agonist-induced phosphorylation were additive for the wild-type AT1-R, but not for the DD mutant receptor. Agonist-induced internalization of the wild-type and DD mutant receptors was similar and was unaltered by coexpression of GRK2K220M. These findings demonstrate that an acidic motif at position 236/237 in the third intracellular loop of the AT1-R is required for optimal Ang II-induced phosphorylation of its carboxyl-terminal tail by GRKs. Furthermore, the properties of the DD mutant receptor suggest that not only Ang II-induced signaling, but also receptor desensitization and internalization, are independent of agonist-induced GRK-mediated phosphorylation of the AT1 receptor.     

Manifold active-state conformations in GPCRs: agonist-activated constitutively active mutant AT1 receptor preferentially couples to Gq compared to the wild-type AT1 receptor

The angiotensin II type I (AT(1)) receptor mediates regulation of blood pressure and water-electrolyte balance by Ang II. Substitution of Gly for Asn(111) of the AT(1) receptor constitutively activates the receptor leading to Gq-coupled IP(3) production independent of Ang II binding. The Ang II-activated conformation of the AT1(N111G) receptor was proposed to be similar to that of the wild-type AT(1) receptor, although, various aspects of the Ang II-induced conformation of this constitutively active mutant receptor have not been systematically studied. Here, we provide evidence that the conformation of the active state of the wild-type and the constitutively active AT(1) receptors are different. Upon Ang II binding an activated conformation of the wild-type AT(1) receptor activates G protein and recruits beta-arrestin. In contrast, the agonist-bound AT1(N111G) mutant receptor preferentially couples to Gq and is inadequate in beta-arrestin recruitment.     

Molecular mechanism underlying inverse agonist of angiotensin II type 1 receptor

To delineate the molecular mechanism underlying the inverse agonist activity of olmesartan, a potent angiotensin II type 1 (AT1) receptor antagonist, we performed binding affinity studies and an inositol phosphate production assay. Binding affinity of olmesartan and its related compounds to wild-type and mutant AT1 receptors demonstrated that interactions between olmesartan and Tyr113, Lys199, His256, and Gln257 in the AT1 receptor were important. The inositol phosphate production assay of olmesartan and related compounds using mutant receptors indicated that the inverse agonist activity required two interactions, that between the hydroxyl group of olmesartan and Tyr113 in the receptor and that between the carboxyl group of olmesartan and Lys199 and His256 in the receptor. Gln257 was found to be important for the interaction with olmesartan but not for the inverse agonist activity. Based on these results, we constructed a model for the interaction between olmesartan and the AT1 receptor. Although the activation of G protein-coupled receptors is initiated by anti-clockwise rotation of transmembrane (TM) III and TM VI followed by changes in the conformation of the receptor, in this model, cooperative interactions between the hydroxyl group and Tyr113 in TM III and between the carboxyl group and His256 in TM VI were essential for the potent inverse agonist activity of olmesartan. We speculate that the specific interaction of olmesartan with these two TMs is essential for stabilizing the AT1 receptor in an inactive conformation. A better understanding of the molecular mechanisms of the inverse agonism could be useful for the development of new G protein-coupled receptor antagonists with inverse agonist activity.     

Identification of the region of AT2 receptor needed for inhibition of the AT1 receptor-mediated inositol 1,4,5-triphosphate generation

Increase in the intracellular inositol triphosphate (IP3) levels in Xenopus oocytes in response to expression and activation of rat angiotensin II (Ang II) receptor AT1 was inhibited by co-expression of rat AT2 receptor. To identify which region of the AT2 was involved in this inhibition, ability of three AT2 mutants to abolish this inhibition was analyzed. Deletion of the C-terminus of the AT2 did not abolish this inhibition. Replacing Ile249 in the third intracellular loop (3rd ICL) of the AT2 with proline, corresponding amino acid in the AT1, in the mutant M6, resulted in slightly reduced affinity to [125I]Ang II (K(d)=0.259 nM), however, did not abolish the inhibition. In contrast, replacing eight more amino acids in the 3rd ICL of the AT2 (at positions 241-244, 250-251 and 255-256) with that of the AT1 in the mutant M8, not only increased the affinity of the AT2 receptor to [125I]Ang II (K(d)=0.038 nM) but also abolished AT2-mediated inhibition. Interestingly, activation of the M8 by Ang II binding also resulted in increase in the intracellular IP(3) levels in oocytes. These results imply that the region of the 3rd ICL of AT2 spanning amino acids 241-256 is sufficient for the AT2-mediated inhibition of AT1-stimulated IP3 generation. Moreover, these nine mutations are also sufficient to render the AT2 with the ability to activate phospholipase C.     

Role of the His273 located in the sixth transmembrane domain of the angiotensin II receptor subtype AT2 in ligand-receptor interaction

Angiotensin II receptor subtypes AT1 and AT2 are proteins with seven transmembrane domain (TMD) topology and share 34% homology. It was shown that His256, located in the sixth TMD of the AT1 receptor, is needed for the agonist activation by the Phe8 side chain of angiotensin II, although replacing this residue with arginine or glutamine did not significantly alter the affinity binding of the receptor. We hypothesized that the His273 located in the sixth transmembrane domain of the AT2 receptor may play a similar role in the functions of the AT2 receptor, although this residue was not identified as a conserved residue in the initial homology comparisions. Therefore, we replaced His273 of the AT2 receptor with arginine or glutamine and analyzed the ligand-binding properties of the mutant receptors using Xenopus oocytes as an expression system. Our results suggested that the AT2 receptor mutants His273Arg and His273 Glu have lost their affinity to [125I-Sar1-Ile8]Ang II, a peptidic ligand that binds both the AT1 and AT2 receptors and to 125I-CGP42112A, a peptidic ligand that binds specifically to the AT2 receptor. Thus, His273 located in the sixth TMD of the AT2 receptor seems to play an important role in determining the binding properties of this receptor. Moreover, these results along with our previous observation that the Lys215 located in the 5th TMD of the AT2 receptor is essential for its high affinity binding to [125I-Sar1-Ile8]Ang II indicate that key amino acids located in the 5th and 6th TMDs of the AT2 receptor are needed for high affinity binding of the AT2 to its ligands.     

Aliphatic amino acids in helix VI of the AT(1) receptor play a relevant role in agonist binding and activity

Angiotensin II (AII) AT(1) receptor mutants with replacements of aliphatic amino acids in the distal region of helix VI and the adjoining region of the third extracellular loop (EC-3) were expressed in Chinese hamster ovary (CHO) cells to determine their role in ligand binding and activation. The triple mutant [L262D, L265D, L268D]AT(1) (L3D) showed a marked reduction in affinity for AII and for non-peptide (losartan) and peptide ([Sar(1)Leu(8) ]AII) antagonists; in functional assays using inositol phosphate (IP) accumulation, the relative potency and the maximum effect of AII were reduced in L3D. Replacement of Leu(268) (in EC-3) and Leu(262) (in the transmembrane domain) by aspartyl residues did not cause significant changes in the receptor's affinity for the ligands and in IP production. In contrast, the point mutation L265D, at helix VI, markedly decreased affinity and ability to stimulate phosphatidylinositol turnover. Molecular modeling of the AT(1) receptor based on a recent crystal structure of rhodopsin, suggests that the side chain of Leu(265) but not that of Leu(262) is facing a cleft between helices V and VI and interacts with the lipid bilayer, thus helping to stabilize the receptor structure near the Lys(199) residue of helix V in the agonist binding site which is necessary for full activity.     

Jak2 acts as both a STAT1 kinase and as a molecular bridge linking STAT1 to the angiotensin II AT1 receptor

Angiotensin II activates the Jak-STAT pathway via the AT(1) receptor. We studied two mutant AT(1) receptors, termed M5 and M6, that contain Y to F substitutions for the tyrosine residues naturally found in the third intracellular loop and the carboxyl terminus. After binding ligand, both the M5 and M6 AT(1) receptors trigger STAT1 tyrosine phosphorylation equivalent to that observed with the wild type receptor, indicating that angiotensin II-mediated phosphorylation of STAT1 is independent of these receptor tyrosine residues. In response to angiotensin II, Jak2 autophosphorylates on tyrosine, and Jak2 and STAT1 physically associate, a process that depends on the SH2 domain of STAT1 in vitro. Evaluation of the wild type, M5, and M6 AT(1) receptors showed that angiotensin II-dependent AT(1) receptor-Jak2-STAT1 complex formation is dependent on catalytically active Jak2, not on the receptor tyrosine residues in the third intracellular loop and carboxyl tail. Immunodepletion of Jak2 virtually eliminated the ligand-dependent binding of STAT1 to the AT(1) receptor. These data indicate that the association of STAT1 with the AT(1) receptor is not strictly bimolecular; it requires Jak2 as both a STAT1 kinase and as a molecular bridge linking STAT1 to the AT(1) receptor.     

Cytoplasmic inositol hexakisphosphate production is sufficient for mediating the Gle1-mRNA export pathway

Production of inositol hexakisphosphate (IP6) by Ipk1, the inositol-1,3,4,5,6-pentakisphosphate 2-kinase, is required for Gle1-mediated mRNA export in Saccharomyces cerevisiae cells. To examine the network of interactions that require IP6 production, an analysis of fitness defects was conducted in mutants harboring both an ipk1 null allele and a mutant allele in genes encoding nucleoporins or transport factors. Enhanced lethality was observed with a specific subset of mutants, including nup42, nup116, nup159, dbp5, and gle2, all of which had been previously connected to Gle1 function. Complementation of the nup116Deltaipk1Delta and nup42Deltaipk1Delta double mutants did not require the Phe-Gly repeat domains in the respective nucleoporins, suggesting that IP6 was acting subsequent to heterogeneous nuclear ribonucleoprotein targeting to the nuclear pore complex. With Nup42 and Nup159 localized exclusively to the nuclear pore complex cytoplasmic side, we speculated that IP6 may regulate a cytoplasmic step in mRNA export. To test this prediction, the spatial requirements for the production of IP6 were investigated. Restriction of Ipk1 to the cytoplasm did not block IP6 production. Moreover, coincident sequestering of both Ipk1 and Mss4 (an enzyme required for phosphatidylinositol 4,5-bisphosphate production) to the cytoplasm also did not block IP6 production. Given that the kinase required for inositol 1,3,4,5,6-pentakisphosphate production (Ipk2) is localized in the nucleus, these results indicated that soluble inositides were diffusing between the nucleus and the cytoplasm. Additionally, the cytoplasmic production of IP6 by plasma membrane-anchored Ipk1 rescued a gle1-2 ipk1-4 synthetic lethal mutant. Thus, cytoplasmic IP6 production is sufficient for mediating the Gle1-mRNA export pathway.     

Role of the third intracellular loop of the angiotensin II receptor subtype AT2 in ligand-receptor interaction

Angiotensin II (Ang II) receptor subtypes AT1 and AT2 share 34% overall homology, but the least homology is in their third intracellular loop (3rd ICL). In an attempt to elucidate the role of the 3rd ICL in determining the similarities and differences in the functions of the AT1 and the AT2 receptors, we generated a chimeric receptor in which the 3rd ICL of the AT2 receptor was replaced with that of the AT1 receptor. Ligand-binding properties and signaling properties of this receptor were assayed by expressing this receptor in Xenopus oocytes. Ligand-binding studies using [125I-Sar1-Ile8] Ang II, a peptidic ligand that binds both the AT1 and the AT2 receptor subtypes, and 125I-CGP42112A, a peptidic ligand that is specific for the AT2 receptor, showed that the chimeric receptor has lost affinity to both ligands. However, IP3 levels of the oocytes expressing the chimeric receptor were comparable to the IP3 levels of the oocytes expressing the AT1 receptor, suggesting that the chimeric receptors could couple to phospholipase C pathway in response to Ang II. We have shown previously that the nature of the amino acid present in the position 215 located in the fifth transmembrane domain (TMD) of the AT2 receptor plays an important role in determining its affinity to different ligands. Our results from the ligand-binding studies of the chimeric receptor further support the idea that the structural organization of the region spanning the 5th TMD and the 3rd ICL of the AT2 receptor has an important role in determining the ligand-binding properties of this receptor.     

Relevant role of Leu265 in helix VI of the angiotensin AT1 receptor in agonist binding and activity

The finding of critical residues for angiotensin II (AII) binding and receptor signalling in helices V and VI led us to assess if, in this region of the receptor, aliphatic side chains might play a role in the agonist-mediated mechanism. Two mutations of the angiotensin AT1 receptor were designed to explore a possible role of a leucine at two positions, Leu265 and Leu268. Thus two mutants, L265D and L268D, were prepared through single substitutions of Leu265, located in the C-terminal region of transmembrane VI (TM-VI), and Leu268, in the adjoining region of the third extracellular loop (EC-3), for an aspartyl residue, and were stably transfected into Chinese hamster ovary (CHO) cells. Ligand-binding experiments and the functional assays determining inositol phosphate (IP) production were performed in these cells expressing these mutants. No significant changes were found in the binding affinity for the ligands, AII, DuP753, and [Sar1Leu8]AII in the mutant L268D. Moreover, the relative potency and the maximum effect on IP production of this mutant were similar to those of the wild-type receptor. In contrast, L265D mutant AT1 receptor, located within the transmembrane domain, markedly decreased binding affinity and ability to stimulate phosphatidylinositol turnover. Our results suggest that the hydrophobic side chain of Leu265, at the C-terminal portion of the AT1's TM-VI, but not Leu268, which belongs to the EC-3 loop, might interact with the AII molecule. On the other side, the aliphatic side chain of Leu265 may be involved in the formation of the ligand binding sites through allosteric effects, thus helping to stabilize the receptor structure around the agonist binding site for full activity.     

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.     

A conserved arginine in the distal third intracellular loop of the mu-opioid receptor is required for G protein activation

In the present study, the functional significance of the intracellular C-terminal loop of the mu-opioid receptor in activating Gi proteins was determined by constructing a C-terminal deletion mutant mu(C delta 45) receptor, which lacks the carboxyl 45 amino acids. When the truncated mu(C delta 45) receptor was stably expressed in human embryonic kidney (HEK) 293 cells, the efficacy and the potency of [D-Ala2,N-Me-Phe4,Gly-ol5]enkephalin (DAMGO), a specific mu-opioid receptor agonist, to inhibit forskolin-stimulated adenylate cyclase activity were not significantly affected. Similar to other G-coupled receptors, the third cytoplasmic loop of the mu-opioid receptor contains conserved basic residues (R276/R277/R280) at the C-terminal segment. Mutating these basic residues to neutral amino acids (L276/M277/L280) greatly impaired the ability of DAMGO to inhibit forskolin-stimulated cyclic AMP formation. Replacing R276/R277 with L276/M277 did not affect the efficacy and potency by which DAMGO inhibits the adenylate cyclase activity. In HEK 293 cells stably expressing mutant (R280L) mu-opioid receptors, the ability of DAMGO to inhibit forskolin-stimulated cyclic AMP production was greatly reduced. These results suggest that the intracellular carboxyl tail of the mu-opioid receptor does not play a significant role in activating Gi proteins and that the arginine residue (R280) at the distal third cytoplasmic loop is required for Gi activation by the mu-opioid receptor.     

Identification of an interaction between the angiotensin II receptor sub-type AT2 and the ErbB3 receptor, a member of the epidermal growth factor receptor family

To identify the proteins that interact and mediate angiotensin II receptor AT2-specific signaling, a random peptide library was screened by yeast-based Two-Hybrid protein-protein interaction assay technique. A peptide that shared significant homology with the amino acids located between the residues Gly-Xaa-Gly-Xaa-Xaa-Gly721 and Lys742, the residues predicted to be important for ATP binding of the ErbB3 and ErbB2 receptors, was identified to be interacting with the AT2 receptor. The interaction between the human ErbB3 receptor and the AT2 receptor was further confirmed using the cytoplasmic domain (amino acids 671-782) of the human ErbB3 receptor. Moreover, an AT2 receptor peptide that spans the amino acids 226-363, (spans the third ICL and carboxy terminal domain) could also interact with the AT2 receptor in a yeast Two-Hybrid protein-protein interaction assay. Studies using mutated and chimeric AT2 receptors showed that replacing the third intracellular loop (ICL) of the AT2 receptor with that of the AT1 abolishes the interaction between the ErbB3 and the AT2 in yeast Two-Hybrid protein-protein interaction assay. Thus the interaction between the AT2 receptor and the ErbB3 receptor seems to require the region spanning the third ICL and carboxy terminus of the AT2 receptor. Since the third ICL of the AT2 receptor is essential for exerting its inhibitory effects on cell growth, possible involvement of this region in the interaction with the cytoplasmic domain of the ErbB3 receptor suggests a novel signaling mechanism for the AT2 receptor mediated inhibition of cell growth. Furthermore, since both the AT2 and the ErbB3 receptors are expressed during fetal development, we propose that the existence of direct interaction between these two receptors may play a role in the regulation of growth during the initial stages of development.     

Endocytosis of the AT1A angiotensin receptor is independent of ubiquitylation of its cytoplasmic serine/threonine-rich region

Agonist-induced internalisation of the rat type 1A (AT(1A)) angiotensin II receptor is associated with phosphorylation of a serine/threonine-rich region in its cytoplasmic tail. In yeast, hyperphosphorylation of the alpha-factor pheromone receptor regulates endocytosis of the receptor by facilitating the monoubiquitylation of its cytoplasmic tail on lysine residues. The role of receptor ubiquitylation in AT(1A) receptor internalisation was evaluated by deletion or replacement of lysine residues in its agonist-sensitive serine/threonine-rich region. Expression of such receptor mutants in CHO cells showed that these modifications had no detectable effect on the angiotensin II-induced endocytosis of the AT(1A) receptor. Furthermore, fusion of ubiquitin in-frame to an internalisation-deficient AT(1A) receptor mutant with a truncated carboxyl-terminal tail did not restore the endocytosis of the resulting chimeric receptor. No impairment of receptor internalisation was observed after substitution of all lysine residues in the serine/threonine-rich region at saturating angiotensin II concentrations, where endocytosis occurs by a beta-arrestin and dynamin independent mechanism. Taken together, these data demonstrate that ubiquitylation of the cytoplasmic serine/threonine-rich region of the AT(1A) receptor on lysine residues is not required for its agonist-induced internalisation, and suggest that endocytosis of mammalian G protein-coupled receptors (GPCRs) occurs by a different mechanism than that of yeast GPCRs.     

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.     

Functional rescue of a defective angiotensin II AT1 receptor mutant by the Mas protooncogene

Earlier studies with Mas protooncogene, a member of the G-protein-coupled receptor family, have proposed this gene to code for a functional AngII receptor, however further results did not confirm this assumption. In this work we investigated the hypothesis that a heterodimeration AT(1)/Mas could result in a functional interaction between both receptors. For this purpose, CHO or COS-7 cells were transfected with the wild-type AT(1) receptor, a non-functional AT(1) receptor double mutant (C18F-K20A) and Mas or with WT/Mas and C18F-K20A/Mas. Cells single-expressing Mas or C18F/K20A did not show any binding for AngII. The co-expression of the wild-type AT(1) receptor and Mas showed a binding profile similar to that observed for the wild-type AT(1) expressed alone. Surprisingly, the co-expression of the double mutant C18F/K20A and Mas evoked a total recovery of the binding affinity for AngII to a level similar to that obtained for the wild-type AT(1). Functional measurements using inositol phosphate and extracellular acidification rate assays also showed a clear recovery of activity for AngII on cells co-expressing the mutant C18F/K20A and Mas. In addition, immunofluorescence analysis localized the AT(1) receptor mainly at the plasma membrane and the mutant C18F-K20A exclusively inside the cells. However, the co-expression of C18F-K20A mutant with the Mas changed the distribution pattern of the mutant, with intense signals at the plasma membrane, comparable to those observed in cells expressing the wild-type AT(1) receptor. These results support the hypothesis that Mas is able to rescue binding and functionality of the defective C18F-K20A mutant by dimerization.     

Analysis of functional domains of angiotensin II type 2 receptor involved in apoptosis.

We previously demonstrated that the intracellular third loop (i3 loop) of angiotensin II type 2 receptor (AT2) plays a key role in mediating the biological functions of this receptor. To determine which residues are important for AT2 signaling, mutated receptors with serial deletions within the i3 loop were stably expressed in PC12 cells. Deletion of residues 240-244 within the intermediate portion of the i3 loop resulted in a complete loss of AT2-mediated apoptosis, inhibition of extracellular signal-regulated kinases (ERK), and SHP-1 activation. In contrast to well characterized heptahelical receptors, the AT2 functions were not affected by deletions of the amino- or carboxyl-terminal portions of the i3 loop. Alanine substitutions further demonstrated that lysine 240, asparagine 242, and serine 243 are key residues for AT2-induced apoptosis, ERK inhibition, and SHP-1 activation. To examine whether a functional link exists between activation of SHP-1 and apoptosis, we used a catalytically inactive SHP-1 mutant and demonstrated that preventing SHP-1 activation strongly attenuates AT2-induced ERK inhibition and apoptosis. Our data demonstrate that the intermediate portion of the i3 loop is important for AT2 function and that SHP-1 is a proximal effector of the AT2 receptor that is implicated in the inhibition of ERKs and in the apoptotic effect of this receptor.     

Chimeric muscarinic cholinergic: beta-adrenergic receptors that activate Gs in response to muscarinic agonists

The M1-muscarinic cholinergic receptor (M1AChR) stimulates the release of inositol phosphates (IPs) but does not activate adenylyl cyclase. The beta-adrenergic receptor (beta-AR) stimulates adenylyl cyclase but has no effect on IP release. Amino acid sequences corresponding to the second (I2) and third (I3) intracellular loops of the turkey erythrocyte beta-AR and a 12-amino acid segment near the N-terminal end of the I3 region were substituted into the corresponding regions of the human M1AChR. Chimeric receptors that contained either the entire I3 loop or the N-terminal dodecapeptide of that loop both mediated the 2-4-fold stimulation of adenylyl cyclase activity in membrane fractions of COS, A293, or Sf9 cells in response to carbachol. These chimeric receptors also retained the ability to stimulate IP release to the same extent as did the M1AChR. In COS cells transfected with the I3 chimeric receptor, the EC50 for carbachol was approximately 7 microM for the stimulation of adenylyl cyclase and approximately 2 microM for the release of IP; M1AChR-mediated IP release displayed an EC50 of approximately 0.2 microM. Substitution of the I2 region of the beta-AR into the M1AChR did not by itself alter selectivity for signaling. However, the I2+I3 and I2+dodecapeptide combined replacements stimulated adenylyl cyclase fully and caused at most 25% of the maximal stimulation of IP release observed with the M1AChR. Thus, a small region in the third cytoplasmic loop can alter the G proteins to which a receptor is coupled, but interaction among loops is evidently involved in fully determining G protein selectivity.     

Threonine 308 within a putative casein kinase 2 site of the cytoplasmic tail of leukotriene B(4) receptor (BLT1) is crucial for ligand-induced,G-protein-coupled receptor-specific kinase 6-mediated desensitization

Desensitization of G-protein-coupled receptors may involve phosphorylation of serine and threonine residues. The leukotriene B(4) (LTB(4)) receptor (BLT1) contains 14 intracellular serines and threonines, 8 of which are part of consensus target sequences for protein kinase C (PKC) or casein kinase 2. In this study, we investigated the importance of PKC and GPCR-specific kinase (GRK) phosphorylation in BLT1 desensitization. Pretreatment of BLT1-transfected COS-7 cells with PKC activators caused a decrease of LTB(4)-induced inositol phosphate (IP) accumulation. This reduction was prevented with the PKC inhibitor, staurosporine, and not observed in cells expressing a BLT1 deletion mutant (G291stop) lacking the cytoplasmic tail. Moreover LTB(4)-induced IP accumulation was significantly inhibited by overexpression of GRK2, GRK5, and especially GRK6, in cells expressing wild type BLT1 but not in those expressing G291stop. GRK6-mediated desensitization correlated with increased phosphorylation of BLT1. The G319stop truncated BLT1 mutant displayed functional characteristics comparable with wild type BLT1 in terms of desensitization by GRK6, but not by PKC. Substitution of Thr(308) within a putative casein kinase 2 site to proline or alanine in the full-length BLT1 receptor prevented most of GRK6-mediated inhibition of LTB(4)-induced IP production but only partially affected LTB(4)-induced BLT1 phosphorylation. Our findings thus suggest that Thr(308) is a major residue involved in GRK6-mediated desensitization of BLT1 signaling.