Hierarchical phosphorylation of delta-opioid receptor regulates agonist-induced receptor desensitization and internalization

Treatment of HEK293 cells expressing the delta-opioid receptor with agonist [d-Pen(2,5)]enkephalin (DPDPE) resulted in the rapid phosphorylation of the receptor. We constructed several mutants of the potential phosphorylation sites (Ser/Thr) at the carboxyl tail of the receptor in order to delineate the receptor phosphorylation sites and the agonist-induced desensitization and internalization. The Ser and Thr were substituted to alanine, and the corresponding mutants were transiently and stably expressed in HEK293 cells. We found that only two residues, i.e. Thr(358) and Ser(363), were phosphorylated, with Ser(363) being critical for the DPDPE-induced phosphorylation of the receptor. Furthermore, using alanine and aspartic acid substitutions, we found that the phosphorylation of the receptor is hierarchical, with Ser(363) as the primary phosphorylation site. Here, we demonstrated that DPDPE-induced rapid receptor desensitization, as measured by adenylyl cyclase activity, and receptor internalization are intimately related to phosphorylation of Thr(358) and Ser(363), with Thr(358) being involved in the receptor internalization.     

Regulation of corticotropin-releasing hormone receptor type 1alpha signaling: structural determinants for G protein-coupled receptor kinase-mediated phosphorylation and agonist-mediated desensitization

Attenuation of CRH receptor type 1 (CRH-R1) signaling activity might involve desensitization and uncoupling of CRH-R1 from intracellular effectors. We investigated the desensitization of native CRH-R in human myometrial cells from pregnant women and recombinant CRH-R1alpha stably overexpressed in human embryonic kidney (HEK) 293 cells. In both cell types, CRH-R1-mediated adenylyl cyclase activation was susceptible to homologous desensitization induced by pretreatment with high concentrations of CRH. Time course studies showed half-maximal desensitization occurring after approximately 40 min of pretreatment and full recovery of CRH-R1alpha functional response within 2 h of removal of CRH pretreatment. In HEK 293 cells, desensitization of CRH-R1alpha was associated with receptor phosphorylation and subsequent endocytosis. To analyze the mechanism leading to CRH-R1alpha desensitization, we overexpressed a truncated beta-arrestin (319-418) and performed coimmunoprecipitation and G protein-coupled receptor kinase (GRK) translocation studies. We found that GRK3 and GRK6 are the main isoforms that interact with CRH-R1alpha, and that recruitment of GRK3 requires Gbetagamma-subunits as well as beta-arrestin. Site-directed mutagenesis of Ser and Thr residues in the CRH-R1alpha C terminus, identified Thr399 as important for GRK-induced receptor phosphorylation and desensitization.We conclude that homologous desensitization of CRH-R1alpha involves the coordinated action of multiple GRK isoforms, Gbeta gamma dimers and beta-arrestin. Based on our identification of key amino acid(s) for GRK-dependent phosphorylation, we demonstrate the importance of the CRH-R1alpha carboxyl tail for regulation of receptor activity.     

Serine 363 Is Required for Nociceptin/Orphanin FQ Opioid Receptor (NOPR) Desensitization,Internalization, and Arrestin Signaling

We determined the role of carboxyl-terminal regulation of NOPR (nociceptin, orphanin FQ receptor) signaling and function. We mutated C-terminal serine and threonine residues and examined their role in NOPR trafficking, homologous desensitization, and arrestin-dependent MAPK signaling. The NOPR agonist, nociceptin, caused robust NOPR-YFP receptor internalization, peaking at 30 min. Mutation of serine 337, 346, and 351, had no effect on NOPR internalization. However, mutation of C-terminal threonine 362, serine 363, and threonine 365 blocked nociceptin-induced internalization of NOPR. Furthermore, point mutation of only Ser-363 was sufficient to block NOPR internalization. Homologous desensitization of NOPR-mediated calcium channel blockade and inhibition of cAMP were also shown to require Ser-363. Additionally, NOPR internalization was absent when GRK3, and Arrestin3 were knocked down using siRNA, but not when GRK2 and Arrestin2 were knocked down. We also found that nociceptin-induced NOPR-mediated JNK but not ERK signaling requires Ser-363, GRK3, and Arrestin3. Dominant-positive Arrestin3 but not Arrestin2 was sufficient to rescue NOPR-S363A internalization and JNK signaling. These findings suggest that NOPR function may be regulated by GRK3 phosphorylation of Ser-363 and Arrestin3 and further demonstrates the complex nature of G-protein-dependent and -independent signaling in opioid receptors.     

Phosphorylation of Ser363, Thr370, and Ser375 residues within the carboxyl tail differentially regulates mu-opioid receptor internalization

Prolonged activation of opioid receptors leads to their phosphorylation, desensitization, internalization, and down-regulation. To elucidate the relationship between mu-opioid receptor (MOR) phosphorylation and the regulation of receptor activity, a series of receptor mutants was constructed in which the 12 Ser/Thr residues of the COOH-terminal portion of the receptor were substituted to Ala, either individually or in combination. All these mutant constructs were stably expressed in human embryonic kidney 293 cells and exhibited similar expression levels and ligand binding properties. Among those 12 Ser/Thr residues, Ser(363), Thr(370), and Ser(375) have been identified as phosphorylation sites. In the absence of the agonist, a basal phosphorylation of Ser(363) and Thr(370) was observed, whereas [d-Ala(2),Me-Phe(4),Gly(5)-ol]enkephalin (DAMGO)-induced receptor phosphorylation occurs at Thr(370) and Ser(375) residues. Furthermore, the role of these phosphorylation sites in regulating the internalization of MOR was investigated. The mutation of Ser(375) to Ala reduced the rate and extent of receptor internalization, whereas mutation of Ser(363) and Thr(370) to Ala accelerated MOR internalization kinetics. The present data show that the basal phosphorylation of MOR could play a role in modulating agonist-induced receptor internalization kinetics. Furthermore, even though mu-receptors and delta-opioid receptors have the same motif encompassing agonist-induced phosphorylation sites, the different agonist-induced internalization properties controlled by these sites suggest differential cellular regulation of these two receptor subtypes.     

Differential phosphorylation paradigms dictate desensitization and internalization of the N-formyl peptide receptor.

Following activation by ligand, most G protein-coupled receptors undergo rapid phosphorylation. This is accompanied by a drastic decrease in the efficacy of continued or repeated stimulation, due to receptor uncoupling from G protein and receptor internalization. Such processing steps have been shown to be absolutely dependent on receptor phosphorylation in the case of the N-formyl peptide receptor (FPR). In this study, we report results that indicate that the mechanisms responsible for desensitization and internalization are distinct. Using site-directed mutagenesis of the serine and threonine residues of the FPR carboxyl terminus, we have characterized regions that differentially regulate these two processes. Whereas substitution of all 11 Ser/Thr residues in the carboxyl terminus prevents both desensitization and internalization, substitution of four Ser/Thr residues between 328-332 blocks desensitization but has no effect on internalization. Similarly, substitution of four Ser/Thr residues between positions 334 and 339 results in a deficit in desensitization but again no decrease in internalization, suggesting that phosphorylation at either site evokes receptor internalization, whereas maximal desensitization requires phosphorylation at both sites. These results also indicate that receptor internalization is not involved in the process of desensitization. Further analysis of the residues between 328-332 revealed that restoration either of Ser(328) and Thr(329) or of Thr(331) and Ser(332) was sufficient to restore desensitization, suggesting that phosphorylation within either of these two sites, in addition to sites between residues 334 and 339, is sufficient to produce desensitization. Taken together, these results indicate that the mechanisms involved in FPR processing (uncoupling from G proteins and internalization) are regulated differentially by phosphorylation at distinct sites within the carboxyl terminus of the FPR. The relevance of this paradigm to other G protein-coupled receptors is discussed.     

Distinct residues in the carboxyl tail mediate agonist-induced desensitization and internalization of the human dopamine D1 receptor.

We have shown in a previous study that desensitization and internalization of the human dopamine D(1) receptor following short-term agonist exposure are mediated by temporally and biochemically distinct mechanisms. In the present study, we have used site-directed mutagenesis to remove potential phosphorylation sites in the third intracellular loop and carboxyl tail of the dopamine D(1) receptor to study these processes. Mutant D(1) receptors were stably transfected into Chinese hamster ovary cells, and kinetic parameters were measured. Mutations of Ser/Thr residues to alanine in the carboxyl tail demonstrated that the single substitution of Thr-360 abolished agonist-induced phosphorylation and desensitization of the receptor. Isolated mutation of the adjacent glutamic acid Glu-359 also abolished agonist-induced phosphorylation and desensitization of the receptor. These data suggest that Thr-360 in conjunction with Glu-359 may comprise a motif necessary for GRK2-mediated phosphorylation and desensitization. Agonist-induced internalization was not affected with mutation of either the Thr-360 or the Glu-359 residues. However, receptors with Ser/Thr residues mutated in the distal carboxyl tail (Thr-446, Thr-439, and Ser-431) failed to internalize in response to agonist activation, but were able to desensitize normally. These results indicate that agonist-induced desensitization and internalization are regulated by separate and distinct serine and threonine residues within the carboxyl tail of the human dopamine D(1) receptor.     

Human complement 5a (C5a) anaphylatoxin receptor (CD88) phosphorylation sites and their specific role in receptor phosphorylation and attenuation of G protein-mediated responses. Desensitization of C5a receptor controls superoxide production but not receptor sequestration in HL-60 cells

Upon agonist binding, the anaphylatoxin human complement 5a receptor (C5aR) has previously been found to be phosphorylated on the six serine residues of its carboxyl-terminal tail (Giannini, E., Brouchon, L., and Boulay, F. (1995) J. Biol. Chem. 270, 19166-19172). To evaluate the precise roles that specific phosphorylation sites may play in receptor signaling, a series of mutants were expressed transiently in COS-7 cells and stably in the physiologically relevant myeloid HL-60 cells. Ser(334) was found to be a key residue that controls receptor phosphorylation. Phosphorylation of either of two serine pairs, namely Ser(332) and Ser(334) or Ser(334) and Ser(338), was critical for the phosphorylation of C5aR and its subsequent desensitization. Full phosphorylation and desensitization of C5aR were obtained when these serines were replaced by aspartic acid residues. The mutation S338A had no marked effect on the agonist-mediated phosphorylation of C5aR, but it allowed a sustained C5a-evoked calcium mobilization in HL-60 cells. These findings and the ability of the S314A/S317A/S327A/S332A mutant receptor to undergo desensitization indicate that the phosphorylation of Ser(334) and Ser(338) is critical and sufficient for C5aR desensitization. The lack of phosphorylation was found to result not only in a sustained calcium mobilization and extracellular signal-regulated kinase 2 activity but also in the enhancement of the C5a-mediated respiratory burst in neutrophil-like HL-60 cells. For instance, the nonphosphorylatable S332A/S334A mutant receptor triggered a 1.8-2-fold higher production of superoxide as compared with the wild-type receptor. Interestingly, although the desensitization of this mutant was defective, it was sequestered with the same time course and the same efficiency as the wild-type receptor. Thus, in myeloid HL-60 cells, desensitization and sequestration of C5aR appear to occur through divergent molecular mechanisms.     

Agonist-dependent desensitization of the kappa opioid receptor by G protein receptor kinase and beta-arrestin.

We used the Xenopus oocyte expression system to examine the regulation of rat kappa opioid receptor (rKOR) function by G protein receptor kinases (GRKs). kappa agonists increased the conductance of G protein-activated inwardly rectifying potassium channels in oocytes co-expressing KOR with Kir3.1 and Kir3.4. In the absence of added GRK and beta-arrestin 2, desensitization of the kappa agonist-induced potassium current was modest. Co-expression of either GRK3 or GRK5 along with beta-arrestin 2 significantly increased the rate of desensitization, whereas addition of either beta-arrestin 2, GRK3, or GRK5 alone had no effect on the KOR desensitization rate. The desensitization was homologous as co-expressed delta opioid receptor-evoked responses were not affected by KOR desensitization. The rate of GRK3/beta-arrestin 2-dependent desensitization was reduced by truncation of the C-terminal 26 amino acids, KOR(Q355Delta). In contrast, substitution of Ala for Ser within the third intracellular loop [KOR(S255A,S260A, S262A)] did not reduce the desensitization rate. Within the C-terminal region, KOR(S369A) substitution significantly attenuated desensitization, whereas the KOR(T363A) and KOR(S356A,T357A) point mutations did not. These results suggest that co-expression of GRK3 or GRK5 and beta-arrestin 2 produced homologous, agonist-induced desensitization of the kappa opioid receptor by a mechanism requiring the phosphorylation of the serine 369 of rKOR.     

Phosphorylation of C3a Receptor at Multiple Sites Mediates Desensitization, β-Arrestin-2 Recruitment and Inhibition of NF-κB Activity in Mast Cells

Background

Phosphorylation of G protein coupled receptors (GPCRs) by G protein coupled receptor kinases (GRKs) and the subsequent recruitment of β-arrestins are important for their desensitization. Using shRNA-mediated gene silencing strategy, we have recently shown that GRK2, GRK3 and β-arrestin-2 promote C3a receptor (C3aR) desensitization in human mast cells. We also demonstrated that β-arrestin-2 provides an inhibitory signal for NF-κB activation. C3aR possesses ten potential phosphorylation sites within its carboxyl terminus but their role on desensitization, β-arrestin recruitment and NF-κB activation has not been determined.

Methodology/Principal Findings

We utilized a site directed mutagenesis approach in transfected HEK293 cells to determine the role of receptor phosphorylation on β-arrestin-2 recruitment and RBL-2H3 cells for functional studies. We found that although Ala substitution of Ser475/479, Thr480/481 residues resulted in 58±3.8% decrease in agonist-induced C3aR phosphorylation there was no change in β-arrestin-2 binding or receptor desensitization. By contrast, Ala substitution of Thr463, Ser465, Thr466 and Ser470 led to 40±1.3% decrease in agonist-induced receptor phosphorylation but this was associated with 74±2.4% decreases in β-arrestin-2 binding, significantly reduced desensitization and enhanced NF-κB activation. Combined mutation of these Ser/Thr residues along with Ser459 (mutant MT7), resulted in complete loss of receptor phosphorylation and β-arrestin-2 binding. RBL-2H3 cells expressing MT7 responded to C3a for greater Ca²⁺ mobilization, degranulation and NF-κB activation when compared to the wild-type receptor. Interestingly, co-expression of MT7 with a constitutively active mutant of β-arrestin (R169E) inhibited C3a-induced degranulation by 28±2.4% and blocked NF-κB activation by 80±2.4%.

Conclusion/Significance

This study demonstrates that although C3a causes phosphorylation of its receptor at multiple sites, Ser459, Thr463, Ser465, Thr466 and Ser470 participate in C3aR desensitization, β-arrestin-2 recruitment and inhibition of NF-κB activity. Furthermore, β-arrestin-2 inhibits C3a-induced NF-κB activation via receptor desensitization-dependent and independent pathways.     

Dual Ser and Thr phosphorylation of CPI-17, an inhibitor of myosin phosphatase, by MYPT-associated kinase

Phosphorylation of CPI-17 and PHI-1 by the MYPT1-associated kinase (M110 kinase) was investigated. M110 kinase is a recently identified serine/threonine kinase with a catalytic domain that is homologous to that of ZIP kinase (ZIPK. GST-rN-ZIPK, a constitutively active GST fusion fragment, phosphorylates CPI-17 (but not PHI-1) to a stoichiometry of 1.7 mol/mol. Phosphoamino acid analysis revealed phosphorylation of both Ser and Thr residues. Phosphorylation sites in CPI-17 were identified as Thr 38 and Ser 12 using Edman sequencing with (32)P release and a point mutant of Thr 38.     

Homologous desensitization of signalling by the alpha (alpha) isoform of the human thromboxane A2 receptor: a specific role for nitric oxide signalling

Thromboxane (TX) A(2) plays a central role in hemostasis, regulating platelet activation status and vascular tone. We have recently established that the TP beta isoform of the human TXA(2) receptor (TP) undergoes rapid, agonist-induced homologous desensitization of signalling largely through a G protein-coupled receptor kinase (GRK) 2/3-dependent mechanism with a lesser role for protein kinase (PK) C. Herein, we investigated the mechanism of desensitization of signalling by the TP alpha isoform. TP alpha undergoes profound agonist-induced desensitization of signalling (intracellular calcium mobilization and inositol 1,4,5 trisphosphate generation) in response to the TXA(2) mimetic U46619 but, unlike that of TP beta, this is independent of GRKs. Similar to TP beta, TP alpha undergoes partial agonist-induced desensitization that occurs through a GF 109203X-sensitive, PKC mechanism where Ser(145) within intracellular domain (IC)(2) represents the key phospho-target. TP alpha also undergoes more profound sustained PKC- and PKG-dependent desensitization where Thr(337) and Ser(331), respectively, within its unique C-tail domain were identified as the phospho-targets. Desensitization was impaired by the nitric oxide synthase (NOS), soluble guanylyl cyclase (sGC) and PKG inhibitors L-NAME, LY 83583 and KT5823, respectively, indicating that homologous desensitization of TP alpha involves nitric oxide generation and signalling. Consistent with this, U46619 led to rapid phosphorylation/activation of endogenous eNOS. Collectively, data herein suggest a mechanism whereby agonist-induced PKC phosphorylation of Ser(145) partially and transiently impairs TP alpha signalling while PKG- and PKC-phosphorylation at both Ser(331) and Thr(337), respectively, within its C-tail domain profoundly desensitizes TP alpha, effectively terminating its signalling. Hence, in addition to the agonist-mediated PKC feedback mechanism, U46619-activation of the NOS/sGC/PKG pathway plays a significant role in inducing homologous desensitization of TP alpha.     

Blockade effects of (Nphe1)Nociceptin(1-13)-NH(2) on anti-nociception induced by intrathecal administration of nociceptin in rats

The present study investigated the roles of the opioid-receptor-like (ORL1) receptor and its endogenous ligand nociceptin on nociception in the spinal cord of rats. Intrathecal administration of 10 nmol of nociceptin produced significant increases in hindpaw withdrawal latencies (HWLs) to thermal and mechanical stimulation. There were no significant changes of average maximum angles in inclined plane tests after intrathecal injection of 10 nmol of nociceptin in rats. The intrathecal nociceptin-induced increases in HWL were antagonized by intrathecal administration of (Nphe1)Nociceptin(1-13)-NH(2), a selective antagonist of ORL1 receptor, in a dose-dependent manner. The results demonstrated that ORL1 receptor is involved in the nociceptin-induced anti-nociceptive effect in the spinal cord of rats.     

Identification of two serine residues essential for agonist-induced 5-HT2A receptor desensitization

5-HT(2A) serotonin receptors represent the principal molecular targets for LSD-like hallucinogens and atypical antipsychotic drugs. It has been proposed that a dysregulation of 5-HT(2A) receptor-mediated signaling may contribute to the pathogenesis of schizophrenia and related diseases. A major mechanism for the attenuation of GPCR signaling following agonist activation typically involves the phosphorylation of serine and/or threonine residues by various kinases. Ser/Thr phosphorylation leads to the binding of accessory proteins and the uncoupling of the G proteins, thereby preventing further signaling. The molecular mechanisms by which 5-HT(2A) receptors are desensitized are unknown, and to date, no residues essential for agonist-mediated desensitization have been identified. Thus, we mutated, individually or in groups, all of the 37 serines and threonines in the cytoplasmic domains of the 5-HT(2A) receptor and assessed the effects of these mutations on agonist-mediated desensitization. We discovered that mutation of two residues, S421 in the C-terminal tail and S188 in the second intracellular loop, to alanine resulted in a significant block of agonist-induced desensitization. Intriguingly, a single-nucleotide polymorphism, of unreported frequency, at the S421 locus has been reported (S421F); the S421F mutation, like the S421A mutation, significantly attenuated agonist-mediated desensitization. Taken together, these findings indicate that the process of agonist-mediated desensitization of 5-HT(2A) receptors requires the presence of two nonconserved serine residues located in distinct intracellular loops.     

Substitution of the autophosphorylation site Thr516 with a negatively charged residue confers constitutive activity to mouse 3-phosphoinositide-dependent protein kinase-1 in cells

3-Phosphoinositide-dependent protein kinase-1 (PDK-1)is a serine/threonine kinase that has been found to phosphorylate and activate several members of the AGC protein kinase family including protein kinase B (Akt), p70 S6 kinase, and protein kinase Czeta. However, the mechanism(s) by which PDK-1 is regulated remains unclear. Here we show that mouse PDK-1 (mPDK-1) undergoes autophosphorylation in vitro on both serine and threonine residues. In addition, we have identified Ser(399) and Thr(516) as the major mPDK-1 autophosphorylation sites in vitro. Furthermore, we have found that these two residues, as well as Ser(244) in the activation loop, are phosphorylated in cells and demonstrated that Ser(244) is a major in vivo phosphorylation site. Abolishment of phosphorylation at Ser(244), but not at Ser(399) or Thr(516), led to a significant decrease of mPDK-1 autophosphorylation and kinase activity in vitro, indicating that autophosphorylation at Ser(399) or Thr(516) is not essential for mPDK-1 autokinase activity. However, overexpression of mPDK-1(T516E), but not of mPDK-1(S244E) or mPDK-1(S399D), in Chinese hamster ovary and HEK293 cells was sufficient to induce Akt phosphorylation at Thr(308) to a level similar to that of insulin stimulation. Furthermore, this increase in phosphorylation was independent of the Pleckstrin homology domain of Akt. Taken together, our results suggest that mPDK-1 undergoes autophosphorylation at multiple sites and that this phosphorylation may be essential for PDK-1 to interact with and phosphorylate its downstream substrates in vivo.     

Deltorphin II-induced rapid desensitization of delta-opioid receptor requires both phosphorylation and internalization of the receptor

Similar to other G protein-coupled receptors, rapid phosphorylation of the delta-opioid receptor in the presence of agonist has been reported. Hence, agonist-induced desensitization of the delta-opioid receptor has been suggested to be via the receptor phosphorylation, arrestin-mediated pathway. However, due to the highly efficient coupling between the delta-opioid receptor and the adenylyl cyclase, the direct correlation between the rates of receptor phosphorylation and receptor desensitization as measured by the adenylyl cyclase activity could not be established. In the current studies, using an ecdysone-inducible expression system to control the delta-opioid receptor levels in HEK293 cells, we could demonstrate that the rate of deltorphin II-induced receptor desensitization is dependent on the receptor level. Only at receptor concentrations 相似文献   

Insulin-induced serine phosphorylation of IRS-2 via ERK1/2 and mTOR: studies on the function of Ser675 and Ser907

The identity of specific serine phosphorylation residues of insulin receptor substrate (IRS)-2 and their impact on insulin signal transduction are largely unknown. Ser(675) and Ser(907) of mouse IRS-2 are adjacent to PI 3-kinase or Grb2 binding domains, respectively. Using monoclonal phosphosite-specific antibodies, we demonstrated the phosphorylation of both serines after stimulation of Fao hepatoma cells with insulin, anisomycin, or phorbol esters. Phosphorylation of both sites was a late and prolonged event during insulin treatment and was also detected in liver tissue of insulin-treated as well as refed mice. Inhibition and siRNA-mediated knockdown of ERK1/2 indicated that the insulin-induced phosphorylation of Ser(907) was ERK dependent. Phosphorylation of Ser(907) did not prevent the insulin-induced association of IRS-2 with Grb2, but phosphorylation of the adjacent Tyr(911) was proved to be crucial in HEK 293 cells expressing IRS-2 Ala mutants. The insulin-induced phosphorylation of Ser(675) was prevented by inhibition and siRNA-mediated knockdown of mTOR but not of p70(S6K1). Mutation of Ser(675) to Ala did not affect downstream insulin signaling but increased the half-life of the protein, suggesting an involvement of phospho-Ser(675) in an accelerated degradation of IRS-2. Moreover, the insulin-induced degradation of IRS-2 was blocked by inhibition of mTOR. We conclude that the two novel insulin-dependent serine phosphorylation sites of IRS-2 were not involved in the regulation of the adjacent PI 3-kinase and Grb2 binding domains but might be implicated in the ERK- and mTOR-mediated negative feedback control.     

Serine 331 is the major site of receptor phosphorylation induced by agents that activate protein kinase G in HEK 293 cells overexpressing thromboxane receptor alpha

Human embryonic kidney (HEK)293 cells stably transfected with the His-tagged thromboxane receptor alpha (TPalpha) was used to study the phosphorylation and desensitization of the receptor induced by 8-bromo-cyclic GMP (8-Br-cGMP), sodium nitroprusside (SNP), or S-nitroso-glutathione (SNG). These agents are known to activate cGMP-dependent protein kinase (PKG). Pretreatment of cells with these agents attenuated significantly agonist I-BOP induced Ca(2+) release. These agents also induced dose-dependent phosphorylation of the TPalpha as demonstrated by increased (32)P-labeling of the receptor from cells prelabeled with (32)Pi. To facilitate the identification of the intracellular domains involved in phosphorylation, glutathione S-transferase (GST)-intracellular domain fusion proteins were used as substrates for the purified PKG. It was found that only the GST-C-terminal tail fusion protein could serve as a substrate for the PKG. To identify the specific serine/threonine residues in the C-terminal tail being phosphorylated, various alanine mutants of these serine/threonine residues were checked for their ability to serve as substrates. It was found that the Ser-331 of the C-terminal tail was primarily involved in the PKG-mediated phosphorylation. That Ser-331 is a predominant site of phosphorylation was supported by in vivo studies in which HEK293 cells expressing the S331A mutant receptor showed little phosphorylation induced by any of the above three agents. Furthermore, HEK293 cells expressing the S331A mutant receptor pretreated with any of the above three agents became responsive to the agonist I-BOP-induced Ca(2+) release. These results indicate that Ser-331 of the TPalpha is the primary site responsible for the phosphorylation and the desensitization of the receptor induced by agents that activate the PKG.     

Beta-arrestin binding to CC chemokine receptor 5 requires multiple C-terminal receptor phosphorylation sites and involves a conserved Asp-Arg-Tyr sequence motif

Agonist binding to the CC chemokine receptor 5 (CCR5) induces the phosphorylation of four distinct serine residues that are located in the CCR5 C terminus. We established a series of clonal RBL-2H3 cell lines expressing CCR5 with alanine mutations of Ser(336), Ser(337), Ser(342), and Ser(349) in various combinations and explored the significance of phosphorylation sites for the ability of the receptor to interact with beta-arrestins and to undergo desensitization and internalization upon ligand binding. Receptor mutants that lack any two phosphorylation sites retained their ability to recruit endogenous beta-arrestins to the cell membrane and were normally sequestered, whereas alanine mutation of any three C-terminal serine residues abolished both beta-arrestin binding and rapid agonist-induced internalization. In contrast, RANTES (regulated on activation normal T cell expressed and secreted) stimulation of a S336A/S349A mutant triggered a sustained calcium response and enhanced granular enzyme release. This mutational analysis implies that CCR5 internalization largely depends on a beta-arrestin-mediated mechanism that requires the presence of any two phosphorylation sites, whereas receptor desensitization is independently regulated by the phosphorylation of distinct serine residues. Surface plasmon resonance analysis further demonstrated that purified beta-arrestin 1 binds to phosphorylated and nonphosphorylated C-tail peptides with similar affinities, suggesting that beta-arrestins use additional receptor sites to discriminate between nonactivated and activated receptors. Surface plasmon resonance analysis revealed beta-arrestin 1 binding to the second intracellular loop of CCR5, which required an intact Asp-Arg-Tyr triplet. These results suggest that a conserved sequence motif within the second intracellular loop of CCR5 that is known to be involved in G protein activation plays a significant role in beta-arrestin binding to CCR5.     

Identification of Ser153 in ICL2 of the gonadotropin-releasing hormone (GnRH) receptor as a phosphorylation-independent site for inhibition of Gq coupling

Type I gonadotropin-releasing hormone (GnRH) receptor (GnRHR) is unique among mammalian G-protein-coupled receptors (GPCRs) in lacking a C-terminal tail, which is involved in desensitization in GPCRs. Therefore, we searched for inhibitory sites in the intracellular loops (ICLs) of the GnRHR. Synthetic peptides corresponding to the three ICLs were inserted into permeabilized alphaT3-1 gonadotrope cells, and GnRH-induced inositol phosphate (InsP) formation was determined. GnRH-induced InsP production was potentiated by ICL2 > ICL3 but not by the ICL1 peptides, suggesting they are acting as decoy peptides. We examined the effects of six peptides in which only one of the Ser or Thr residues was substituted with Ala or Glu. Only substitution of Ser153 with Ala or Glu ablated the potentiating effect upon GnRH-induced InsP elevation. ERK activation was enhanced, and the rate of GnRH-induced InsP formation was about 6.5-fold higher in the first 10 min in COS-1 cells that were transfected with mutants of the GnRHR in which the ICL2 Ser/Thr residues (Ser151, Ser153, and Thr142) or only Ser153 was mutated to Ala as compared with the wild type GnRHR. The data indicate that ICL2 harbors an inhibitory domain, such that exogenous ICL2 peptide serves as a decoy for the inhibitory site (Ser153) of the GnRHR, thus enabling further activation. GnRH does not induce receptor phosphorylation in alphaT3-1 cells. Because the phosphomimetic ICL2-S153E peptide did not mimic the stimulatory effect of the ICL2 peptide, the inhibitory effect of Ser153 operates through a phosphorylation-independent mechanism.