Phosphorylation of the Gq/11-coupled m3-muscarinic receptor is involved in receptor activation of the ERK-1/2 mitogen-activated protein kinase pathway

We investigated the role played by agonist-mediated phosphorylation of the G(q/11)-coupled M(3)-muscarinic receptor in the mechanism of activation of the mitogen-activated protein kinase pathway, ERK-1/2, in transfected Chinese hamster ovary cells. A mutant of the M(3)-muscarinic receptor, where residues Lys(370)-Ser(425) of the third intracellular loop had been deleted, showed a reduced ability to activate the ERK-1/2 pathway. This reduction was evident despite the fact that the receptor was able to couple efficiently to the phospholipase C second messenger pathway. Importantly, the ERK-1/2 responses to both the wild-type M(3)-muscarinic receptor and DeltaLys(370)-Ser(425) receptor mutant were dependent on the activity of protein kinase C. Our results, therefore, indicate the existence of two mechanistic components to the ERK-1/2 response, which appear to act in concert. First, the activation of protein kinase C through the diacylglycerol arm of the phospholipase C signaling pathway and a second component, absent in the DeltaLys(370)-Ser(425) receptor mutant, that is independent of the phospholipase C signaling pathway. The reduced ability of the DeltaLys(370)-Ser(425) receptor mutant to activate the ERK-1/2 pathway correlated with an approximately 80% decrease in the ability of the receptor to undergo agonist-mediated phosphorylation. Furthermore, we have previously shown that M(3)-muscarinic receptor phosphorylation can be inhibited by a dominant negative mutant of casein kinase 1alpha and by expression of a peptide corresponding to the third intracellular loop of the M(3)-muscarinic receptor. Expression of these inhibitors of receptor phosphorylation reduced the wild-type M(3)-muscarinic receptor ERK-1/2 response. We conclude that phosphorylation of the M(3)-muscarinic receptor on sites in the third intracellular loop by casein kinase 1alpha contributes to the mechanism of receptor activation of ERK-1/2 by working in concert with the diacylglycerol/PKC arm of the phospholipase C signaling pathway.     

Phosphorylation and regulation of a G protein-coupled receptor by protein kinase CK2

We demonstrate a role for protein kinase casein kinase 2 (CK2) in the phosphorylation and regulation of the M3-muscarinic receptor in transfected cells and cerebellar granule neurons. On agonist occupation, specific subsets of receptor phosphoacceptor sites (which include the SASSDEED motif in the third intracellular loop) are phosphorylated by CK2. Receptor phosphorylation mediated by CK2 specifically regulates receptor coupling to the Jun-kinase pathway. Importantly, other phosphorylation-dependent receptor processes are regulated by kinases distinct from CK2. We conclude that G protein-coupled receptors (GPCRs) can be phosphorylated in an agonist-dependent fashion by protein kinases from a diverse range of kinase families, not just the GPCR kinases, and that receptor phosphorylation by a defined kinase determines a specific signalling outcome. Furthermore, we demonstrate that the M3-muscarinic receptor can be differentially phosphorylated in different cell types, indicating that phosphorylation is a flexible regulatory process where the sites that are phosphorylated, and hence the signalling outcome, are dependent on the cell type in which the receptor is expressed.     

Acute agonist-mediated desensitization of the human alpha 1a-adrenergic receptor is primarily independent of carboxyl terminus regulation: implications for regulation of alpha 1aAR splice variants.

Despite important roles in myocardial hypertrophy and benign prostatic hyperplasia, little is known about acute effects of agonist stimulation on alpha(1a)-adrenergic receptor (alpha(1a)AR) signaling and function. Regulatory mechanisms are likely complex since 12 distinct human alpha(1a)AR carboxyl-terminal splice variants have been isolated. After determining the predominance of the alpha(1a-1)AR isoform in human heart and prostate, we stably expressed an epitope-tagged alpha(1a-1)AR cDNA in rat-1 fibroblasts and subsequently examined regulation of signaling, phosphorylation, and internalization of the receptor. Human alpha(1a)AR-mediated inositol phosphate signaling is acutely desensitized in response to both agonist and phorbol 12-myristate 13-acetate (PMA) exposure. Concurrent with desensitization, alpha(1a)ARs in (32)P(i)-labeled cells are rapidly phosphorylated in response to both NE and PMA stimulation. Despite the ability of PKC to desensitize alpha(1a)ARs when directly activated with PMA, inhibitors of PKC have no effect on agonist-mediated desensitization. In contrast, involvement of GRK kinases is suggested by the ability of GRK2 to desensitize alpha(1a)ARs. Internalization of cell surface alpha(1a)ARs also occurs in response to agonist stimulation (but not PKC activation), but is initiated more slowly than receptor desensitization. Significantly, deletion of the alpha(1a)AR carboxyl terminus has no effect on receptor internalization or either agonist-induced or GRK-mediated receptor desensitization. Because mechanisms underlying acute agonist-mediated regulation of human alpha(1a)ARs are primarily independent of the carboxyl terminus, they may be common to all functional alpha(1a)AR isoforms.     

Insulin-like growth factor-I induces alpha(1B)-adrenergic receptor phosphorylation through G beta gamma and epidermal growth factor receptor transactivation

IGF-I induces alpha(1B)-adrenoceptor (alpha(1B)-AR) phosphorylation. The effect of IGF-I was rapid and transient, reaching near-maximal values at 10 min and decreasing after 30 min; it was observed at low IGF-I concentrations (EC(50) approximately 10 ng/ml) and was associated to receptor desensitization as evidenced by a decreased alpha(1B)-adrenergic effect on intracellular calcium and production of inositol phosphates. The effect of IGF-I was markedly decreased in cells treated with pertussis toxin suggesting involvement of pertussis toxin-sensitive G proteins. Transfection of the carboxyl terminus of the beta-adrenergic receptor kinase or the Deltap85 mutant of phosphoinositide 3-kinase (PI3K) markedly decreased the alpha(1B)-AR phosphorylation induced by IGF-I without decreasing the receptor phosphorylation induced by noradrenaline. Inhibitors of PI3K and protein kinase C blocked IGF-I-induced alpha(1B)-AR phosphorylation. In addition, it was observed that AG1478, an inhibitor of the epidermal growth factor (EGF) receptor kinase, and BB-94, a metalloproteinase inhibitor, also diminished IGF-I-induced adrenoceptor phosphorylation. The data clearly show that IGF-I triggers a complex signaling pathway, which leads to the phosphorylation and desensitization of a serpentine G protein-coupled receptor, suggesting the following hypothetical model: 1) stimulation of IGF-I receptors activate pertussis toxin-sensitive G proteins; 2) the growth factor action activates metalloproteinases, which catalyze heparin binding-EGF shedding, and transactivation of EGF receptors, and 3) dissociated Gbetagamma subunits and phosphotyrosine residues seem to trigger PI3K activity, which leads to activation of protein kinase C, resulting in alpha(1B)-AR phosphorylation and desensitization.     

Termination of protease-activated receptor-1 signaling by beta-arrestins is independent of receptor phosphorylation

Protease-activated receptor 1 (PAR1), a G protein-coupled receptor (GPCR) for thrombin, is the prototypic member of a family of protease-activated receptors. PAR1 is irreversibly proteolytically activated; thus, the magnitude and duration of thrombin cellular responses are determined primarily by mechanisms responsible for termination of receptor signaling. Both phosphorylation and beta-arrestins contribute to rapid desensitization of PAR1 signaling. However, the relative contribution of each of these pathways to the termination of PAR1 signaling is not known. Co-expression of PAR1 with beta-arrestin 1 (betaarr1) in COS-7 cells resulted in a marked inhibition of PAR1 signaling, whereas beta-arrestin 2 (betaarr2) was essentially inactive. Strikingly, signaling by a PAR1 cytoplasmic tail mutant defective in agonist-induced phosphorylation was also attenuated more effectively by betaarr1 compared with betaarr2. In contrast, both beta-arrestin isoforms were equally effective at desensitizing the substance P receptor, a classic reversibly activated GPCR. PAR1 coimmunoprecipitated betaarr1 in an agonist-dependent manner, whereas betaarr2 association was virtually undetectable. Remarkably, betaarr1 also interacted with phosphorylation defective PAR1 mutant, whereas betaarr2 did not. Moreover, constitutively active beta-arrestin mutants, betaarr1 R169E and betaarr2 R170E, that bind to activated receptor independent of phosphorylation failed to enhance either wild type or mutant PAR1 desensitization compared with normal versions of these proteins. In contrast, beta-arrestin mutants displayed enhanced activity at desensitizing the serotonin 5-hydroxytryptamine(2A) receptor. Taken together, these results suggest that, in addition to PAR1 cytoplasmic tail phosphorylation itself, beta-arrestin binding independent of phosphorylation promotes desensitization of PAR1 signaling. These findings reveal a new level of complexity in the regulation of protease-activated GPCR signaling.     

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.     

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.     

Serine 232 of the alpha(2A)-adrenergic receptor is a protein kinase C-sensitive effector coupling switch.

alpha(2)-adrenergic receptors (alpha(2)AR) couple to multiple effectors including adenylyl cyclase and phospholipase C. We hypothesized that signaling selectivity to these effectors is dynamically directed by kinase-sensitive domains within the third intracellular loop of the receptor. Substitution of Ala for Ser232, which is in the N-terminal region of this loop in the alpha(2A)AR, resulted in a receptor that was markedly uncoupled ( approximately 82% impairment) from stimulation of inositol phosphate accumulation while the capacity to inhibit adenylyl cyclase remained relatively intact. In S232A alpha(2A)AR transfected cell membranes, agonist-promoted [(35)S]GTPgammaS binding was reduced by approximately 50%. Coexpression of modified G proteins rendered insensitive to pertussis toxin revealed that the S232A receptor was uncoupled from both G(i) and G(o). S232 is a potential PKC phosphorylation site, and whole cell phosphorylation studies showed that the mutant had depressed phosphorylation compared to wild type (1.3- vs 2.1-fold/basal). Consistent with S232 directing coupling to phospholipase C, PMA exposure resulted in approximately 67% desensitization of agonist-promoted inositol phosphate accumulation without significantly affecting inhibition of adenylyl cyclase. The dominant effect of mutation or phosphorylation at this site on inositol phosphate as compared to cAMP signaling was found to most likely be due to the low efficiency of signal transduction via phospholipase C vs adenylyl cyclase. Taken together, these results indicate that S232 acts as a selective, PKC-sensitive, modulator of effector coupling of the alpha(2A)AR to inositol phosphate stimulation. This represents one mechanism by which cells route stimuli directed to multifunctional receptors to selected effectors so as to attain finely targeted signaling.     

The carboxyl terminus of the hamster beta-adrenergic receptor expressed in mouse L cells is not required for receptor sequestration

The structural basis for agonist-mediated sequestration and desensitization of the beta-adrenergic receptor (beta AR) was examined by oligonucleotide-directed mutagenesis of the hamster beta AR gene and expression of the mutant genes in mouse L cells. Treatment of these cells with the agonist isoproterenol corresponded to a desensitization of beta AR activity. A mutant receptor that bound agonist but did not couple to adenylate cyclase showed a dramatically reduced sequestration response to agonist stimulation. In contrast, beta AR mutants in which the C-terminus was truncated and/or in which two regions that have been proposed as phosphorylation substrates for cAMP-dependent protein kinase were removed showed normal sequestration responses. These results demonstrate that agonist-mediated sequestration of the beta AR can occur in the absence of the C-terminus of the protein and reveal a strong correlation between effective coupling to Gs and sequestration.     

Roles of phosphorylation-dependent and -independent mechanisms in the regulation of M1 muscarinic acetylcholine receptors by G protein-coupled receptor kinase 2 in hippocampal neurons

When co-expressed with the inositol 1,4,5-trisphosphate biosensor eGFP-PH(PLC delta), G protein-coupled receptor kinase 2 (GRK2) can suppress M1 muscarinic acetylcholine (mACh) receptor-mediated phospholipase C signaling in hippocampal neurons through a phosphorylation-independent mechanism, most likely involving the direct binding of the RGS homology domain of GRK2 to G alpha(q/11). To define the importance of this mechanism in comparison with classical, phosphorylation-dependent receptor regulation by GRKs, we have examined M1 mACh receptor signaling in hippocampal neurons following depletion of GRK2 and also in the presence of non-G alpha(q/11)-binding GRK2 mutants. Depletion of neuronal GRK2 using an antisense strategy almost completely inhibited M1 mACh receptor desensitization without enhancing acute agonist-stimulated phospholipase C activity. By stimulating neurons with a submaximal agonist concentration before (R1) and after (R2) a period of exposure to a maximal agonist concentration, an index (R2/R1) of agonist-induced desensitization of signaling could be obtained. Co-transfection of neurons with either a non-G alpha(q/11)-binding (D110A) GRK2 mutant or the catalytically inactive (D110A,K220R)GRK2 did not suppress acute M1 mACh receptor-stimulated inositol 1,4,5-trisphosphate production. However, using the desensitization (R2/R1) protocol, it could be shown that expression of (D110A)GRK2 enhanced, whereas (D110A,K220R)GRK2 inhibited, agonist-induced M1 mACh receptor desensitization. In Chinese hamster ovary cells, the loss of G alpha(q/11) binding did not affect the ability of the (D110A)GRK2 mutant to phosphorylate M1 mACh receptors, whereas expression of (D110A,K220R)GRK2 had no effect on receptor phosphorylation. These data indicate that in hippocampal neurons endogenous GRK2 is a key regulator of M1 mACh receptor signaling and that the regulatory process involves both phosphorylation-dependent and -independent mechanisms.     

Desensitization of the mouse thromboxane A2 receptor (TP) by G protein-coupled receptor kinases (Grks)

GRKs play a key role in regulating G protein-coupled receptor (GPCR) responsiveness. To investigate the role of GRKs in desensitization of TP, we replaced threonines with favorable phosphorylation motifs for GRKs (positions 226 and 230) with alanine. Mutant and wild-type receptors were expressed in cell culture models and clones expressing similar numbers of receptors were studied. We found that: (1) affinity and specificity of thromboxane A2 (TxA2) binding to mutant TP were identical to the wild-type, (2) replacement of threonines 226 and 230 with alanines delayed the onset of agonist-induced desensitization, and (3) inhibition of endogenous GRK activity with a dominant-negative construct inhibited agonist-induced phosphorylation and enhanced responsiveness of wild-type TP but had little effect on responsiveness of the receptor mutant. These data are consistent with the notion that GRKs contribute to desensitization of TP.     

Glycogen Synthase Kinase-3 modulates α1A-adrenergic receptor action and regulation

The possibility that glycogen synthase kinase 3 (GSK3) could modulate α_1A-adrenergic receptor (α_1A-AR) function and regulation was tested employing LNCaP and HEK293 cells transfected to express the enhanced green fluorescent protein-tagged human α_1A-AR. Receptor phosphorylation and internalization, intracellular free calcium, α_1A-AR-GSK3 colocalization, and coimmunoprecipitation were studied. The effects of the pharmacological GSK3 inhibitor, SB-216763, and the coexpression of a dominant-negative mutant of this kinase, as well as the signaling, desensitization, and internalization of receptors with S229, S258, S352, and S381 substitutions for alanine or aspartate, were also determined. SB-216763 inhibited agonist- and phorbol myristate acetate (PMA)-mediated α_1A-AR phosphorylation, reduced oxymetazoline-induced desensitization, and magnified that induced by PMA. Agonists and PMA increased receptor-GSK3 colocalization and coimmunoprecipitation. Expression of a dominant-negative GSK3 mutant reduced agonist- but not PMA-induced receptor internalization. α_1A-AR with the GSK3 putative target sites mutated to alanine exhibited reduced phosphorylation and internalization in response to agonists and increased PMA-induced desensitization. Agonist-induced, but not PMA-induced, receptor-β arrestin intracellular colocalization was diminished in cells expressing the GSK3 putative target sites mutated to alanine. Our data indicated that GSK3 exerts a dual action on α_1A-AR participating in agonist-mediated desensitization and internalization and avoiding PMA-induced desensitization.     

Endogenous G protein-coupled receptor kinase 6 Regulates M3 muscarinic acetylcholine receptor phosphorylation and desensitization in human SH-SY5Y neuroblastoma cells

We have previously shown that overexpression of G protein-coupled receptor kinase 6 (GRK6) enhanced the phosphorylation and desensitization of the endogenously expressed M(3) muscarinic acetylcholine (mACh) receptor in human SH-SY5Y neuroblastoma cells. In this study we have examined the potential role of endogenous GRK6 in the regulation of M(3) mACh receptor by blocking its action through the introduction of a kinase-dead, dominant-negative GRK6 ((K215R)GRK6). (K215R)GRK6 expression inhibited methacholine-stimulated M(3) mACh receptor phosphorylation by 50% compared with plasmid transfected control cells. Guanosine-5'-O-(3-[(35)S]thio)triphosphate binding and immunoprecipitation studies, conducted after agonist pretreatment (3 min), indicated that M(3) mACh receptor-G alpha(q/11) uncoupling was attenuated by 50% in cells expressing (K215R)GRK6 when compared with control cells. In contrast, expression of the related dominant-negative kinase (K215R)GRK5 had no effect on M(3) mACh receptor phosphorylation or uncoupling. Time course studies also showed that agonist-stimulated [(3)H]inositol phosphate accumulations were more sustained in cells expressing (K215R)GRK6 compared with control and (K215R)GRK5-expressing cells, whereas (K215R)GRK6 expression had no effect on the phospholipase C response to direct stimulation of G proteins with AlF(4)(-). The ability of (K215R)GRK6 to inhibit agonist-mediated M(3) mACh receptor phosphorylation and G protein uncoupling suggests that endogenous GRK6 mediates, at least in part, M(3) mACh receptor desensitization in the SH-SY5Y cell line.     

Phospholipase D2 modulates agonist-induced mu-opioid receptor desensitization and resensitization

Receptor phosphorylation, arrestin binding, uncoupling from G protein and subsequent endocytosis have been implicated in G protein-coupled receptor desensitization after chronic agonist exposure. In search of proteins regulating the mu-opioid receptor endocytosis, we have recently established that activation of phospholipase D (PLD)2 is required for agonist-induced mu-opioid receptor endocytosis. In this study, we determined the effect of PLD2 activity on the desensitization and resensitization rate of the mu-opioid receptor. We clearly demonstrated that inhibition of PLD2-mediated phosphatidic acid formation by alcohol (1-butanol or ethanol) or overexpression of a dominant negative mutant of PLD2 prevented agonist-mediated endocytosis and resulted in a faster desensitization rate of the mu-opioid receptor after chronic (D-Ala2, Me Phe4, Glyol5)enkephalin treatment in human embryonic kidney 293 cells. Moreover, inhibition of PLD2 activity led to an impairment of the resensitization rate of the mu-opioid receptor. In summary, our data strongly suggest that PLD2 is a modulator of agonist-induced endocytosis, desensitization and resensitization of the mu-opioid receptor.     

Beta-arrestin1 and beta-arrestin2 are differentially required for phosphorylation-dependent and -independent internalization of delta-opioid receptors

Beta-arrestins are key negative regulators and scaffolds of G protein-coupled receptor (GPCR) signalling. Beta-arrestin1 and beta-arrestin2 preferentially bind to the phosphorylated GPCRs in response to agonist stimulation, resulting in receptor internalization and desensitization. The critical roles of GPCR kinases (GRKs)-catalyzed receptor phosphorylation and interaction of beta-arrestins with the phosphorylated receptor in receptor internalization are well established. However, emerging evidence suggests that an agonist-stimulated internalization mechanism that is independent of receptor phosphorylation may also be employed in some cases, although the molecular mechanism for the phosphorylation-independent GPCR internalization is not clear. The current study investigated the role of receptor phosphorylation and the involvement of different beta-arrestin subtypes in agonist-induced delta-opioid receptor (DOR) internalization in HEK293 cells. Results from flow cytometry, fluorescence microscopy, and surface biotin labelling experiments showed that elimination of agonist-induced DOR phosphorylation by mutation GRK binding or phosphorylation sites only partially blocked agonist-induced receptor internalization, indicating the presence of an agonist-induced, GRK-independent mechanism for DOR internalization. Fluorescence and co-immunoprecipitation studies indicated that both the wild-type DOR and the phosphorylation-deficient mutant receptor could bind and recruit beta-arrestin1 and beta-arrestin2 to the plasma membrane in an agonist-stimulated manner. Furthermore, internalization of both the wild-type and phosphorylation-deficient receptors was increased by overexpression of either type of beta-arrestins and blocked by dominant-negative mutants of beta-arrestin-mediated internalization, demonstrating that both phosphorylation-dependent and -independent internalization require beta-arrestin. Moreover, double-stranded RNA-mediated interference experiments showed that either beta-arrestin1 or beta-arrestin2 subtype-specific RNAi only partially inhibited agonist-induced internalization of the wild-type DOR. However, agonist-induced internalization of the phosphorylation-deficient DOR was not affected by beta-arrestin1-specific RNAi but was blocked by RNAi against beta-arrestin2 subtype. These data indicate that endogenous beta-arrestin1 functions exclusively in the phosphorylation-dependent receptor internalization, whereas endogenous beta-arrestin2, but not beta-arrestin1, is required for the phosphorylation-independent receptor internalization. These results thus provide the first evidence of different requirement for beta-arrestin isoforms in the agonist induced phosphorylation-dependent and -independent GPCR internalization.     

Regulation of human melanocortin 1 receptor signaling and trafficking by Thr-308 and Ser-316 and its alteration in variant alleles associated with red hair and skin cancer

The melanocortin 1 receptor (MC1R), a G protein-coupled receptor (GPCR) positively coupled to adenylyl cyclase, is a key regulator of melanocyte proliferation and differentiation and a determinant of pigmentation, skin phototype, and skin cancer risk. MC1R activation stimulates melanogenesis and increases the ratio of black, strongly photoprotective eumelanins to yellowish and poorly photoprotective pheomelanin pigments. Desensitization and internalization are key regulatory mechanisms of GPCR signaling. Agonist-induced desensitization usually depends on phosphorylation by a GPCR kinase (GRK) followed by receptor internalization in endocytic vesicles. We have shown that MC1R desensitization is mediated by two GRKs expressed in melanocytes and melanoma cells, GRK2 and GRK6. Here we show that in contrast with this dual specificity for desensitization, GRK6 but not GRK2 mediated MC1R internalization. Mutagenesis studies suggested that the targets of GRK6 are two residues located in the MC1R cytosolic C terminus, Thr-308 and Ser-316. A T308D/S316D mutant mimicking their phosphorylated state was constitutively desensitized and associated with endosomes, whereas a T308A/S316A mutant was resistant to desensitization and internalization. We studied the desensitization and internalization of three variant MC1R forms associated with red hair and increased skin cancer risk: R151C, R160W, and D294H. These variants showed a less efficient desensitization. Moreover, D294H was resistant to internalization, thus accounting for its abnormally high surface expression. Co-expression of variant and wild type MC1R modified its desensitization and internalization behavior. These data suggest that MC1R might be regulated by novel mechanisms including differential effects of GRKs and altered desensitization rates of certain allelic combinations.     

Human substance P receptor lacking the C-terminal domain remains competent to desensitize and internalize

Substance P receptor (SPR) and its naturally occurring splice-variant, lacking the C-terminal tail, are found in brain and spinal cord. Whether C-terminally truncated SPR desensitizes like full-length SPR is controversial. We used a multivaried approach to determine whether human SPR (hSPR) and a C-terminally truncated mutant, hSPRDelta325, differ in their desensitization and internalization. In HEK-293 cells expressing either hSPRDelta325 or hSPR, SP-induced desensitization of the two receptors was similar when measured by inositol triphosphate accumulation or by transient translocation of coexpressed PKCbetaII-GFP to the plasma membrane. Moreover, translocation of beta-arrestin 1 or 2-GFP (betaarr1-GFP or betaarr2-GFP) to the plasma membrane, and receptor internalization were also similar. However, hSPR and hSPRDelta325 differ in their phosphorylation and in their ability to form beta-arrestin-containing endocytic vesicles. Unlike hSPR, hSPRDelta325 is not phosphorylated to a detectable level in intact HEK293 cells, and whereas hSPR forms vesicles containing either betaarr1-GFP or betaarr2-GFP, hSPRDelta325 does not form any vesicles with betaarr1-GFP, and forms fewer vesicles with betaarr2-GFP. We conclude that truncated hSPR undergoes agonist-dependent desensitization and internalization without detectable receptor phosphorylation.     

Activation of the mitogen-activated protein kinase pathway by a Gq/11-coupled muscarinic receptor is independent of receptor internalization

A number of recent studies have demonstrated an essential role for receptor endocytosis in the activation of the mitogen-activated protein (MAP) kinases, Erk-1 and Erk-2 (extracellular activated protein kinases 1 and 2), by growth factor receptors and the G-protein coupled beta2-adrenergic receptor. Because ligand-mediated receptor endocytosis and activation of the MAP kinase pathway are common phenomena among G-protein coupled receptors, it has been suggested that the essential role of endocytosis in MAP kinase activation identified for the beta2-adrenergic receptor may be universal for all G-protein coupled receptors (Daaka,Y., Luttrell, L. M., Ahn, S., Della Rocca, G. J., Ferguson, S. S. G., Caron, M. G., and Lefkowitz, R. J. (1998) J. Biol. Chem. 273, 685-688). We tested this hypothesis using the Gq/11-coupled m3-muscarinic receptor expressed in Chinese hamster ovary cells and an m3-muscarinic receptor mutant that does not undergo endocytosis. We demonstrate that inhibition of endocytosis by concanavalin A and cytochalasin D does not affect the ability of the wild type m3-muscarinic receptor to activate Erk-1/2. Furthermore, the mutant m3-muscarinic receptor that is unable to undergo endocytosis, activates the MAP kinase pathway in an identical manner to the wild type receptor. We conclude that receptor endocytosis is not universally essential for MAP kinase activation by G-protein coupled receptors. We discuss the possibility that the differential roles played by endocytosis in MAP kinase activation between various receptor subtypes may be linked to the mechanism of upstream activation of Raf-1.     

Untangling ligand induced activation and desensitization of G-protein-coupled receptors

Long-term treatment with a drug to a G-protein-coupled receptor (GPCR) often leads to receptor-mediated desensitization, limiting the therapeutic lifetime of the drug. To better understand how this therapeutic window might be controlled, we created a mechanistic Monte Carlo model of the early steps in GPCR signaling and desensitization. Using this model we found that the rates of G-protein activation and receptor phosphorylation can be partially decoupled by varying the drug-receptor dissociation rate constant, k(off), and the drug's efficacy, alpha. The maximum ratio of G-protein activation to receptor phosphorylation (GARP) was found for drugs with an intermediate k(off) value and small alpha-value. Changes to the cellular environment, such as changes in the diffusivity of membrane molecules and the G-protein inactivation rate constant, affected the GARP value of a drug but did not change the characteristic shape of the GARP curve. These model results are examined in light of experimental data for a number of GPCRs and are found to be in good agreement, lending support to the idea that the desensitization properties of a drug might be tailored to suit a specific application.     

Phosphorylation and Desensitization of the Neurokinin-1 Receptor Expressed in Epithelial Cells

A rat kidney epithelial cell line expressing the rat neurokinin-1 receptor (NK-1 R) was used to investigate the relationship between receptor phosphorylation and desensitization. Substance P (SP) maximally stimulated cellular inositol 1,4,5-trisphosphate (IP3) production 14-fold within 3 s, after which cellular IP3 levels rapidly diminished to near basal levels in the continuing presence of SP. SP also caused concentration-dependent phosphorylation of the NK-1R, and this effect was blocked by a receptor antagonist. Stimulation with 100 nM SP for as little as 2 s resulted in 90% desensitization of the receptor to restimulation by SP, and near-maximal receptor phosphorylation was observed at 5 s. Receptor desensitization was not affected by agents that affect protein kinase A. Phorbol 12-myristate 13-acetate (PMA) also caused phosphorylation and desensitization of the receptor but with slower kinetics and to a lesser extent than SP. PMA- but not SP-induced NK-1 R desensitization and phosphorylation were abolished by the protein kinase C inhibitor bisindolylmaleimide 1. The concentration-response curves for SP-stimulated IP3 signaling and desensitization were similar, but the curve for NK-1R phosphorylation was shifted to the right and was steeper, suggesting that the relationship between desensitization and phosphorylation is complex. These results show that both rapid homologous and rapid heterologous NK-1R desensitizations may be mediated by receptor phosphorylation but occur via distinct mechanisms with different kinetics and efficacies.