Site-specific Phosphorylation of CXCR4 Is Dynamically Regulated by Multiple Kinases and Results in Differential Modulation of CXCR4 Signaling

The chemokine receptor CXCR4 is a widely expressed G protein-coupled receptor that has been implicated in a number of diseases including human immunodeficiency virus, cancer, and WHIM syndrome, with the latter two involving dysregulation of CXCR4 signaling. To better understand the role of phosphorylation in regulating CXCR4 signaling, tandem mass spectrometry and phospho-specific antibodies were used to identify sites of agonist-promoted phosphorylation. These studies demonstrated that Ser-321, Ser-324, Ser-325, Ser-330, Ser-339, and two sites between Ser-346 and Ser-352 were phosphorylated in HEK293 cells. We show that Ser-324/5 was rapidly phosphorylated by protein kinase C and G protein-coupled receptor kinase 6 (GRK6) upon CXCL12 treatment, whereas Ser-339 was specifically and rapidly phosphorylated by GRK6. Ser-330 was also phosphorylated by GRK6, albeit with slower kinetics. Similar results were observed in human astroglia cells, where endogenous CXCR4 was rapidly phosphorylated on Ser-324/5 by protein kinase C after CXCL12 treatment, whereas Ser-330 was slowly phosphorylated. Analysis of CXCR4 signaling in HEK293 cells revealed that calcium mobilization was primarily negatively regulated by GRK2, GRK6, and arrestin3, whereas GRK3, GRK6, and arrestin2 played a primary role in positively regulating ERK1/2 activation. In contrast, GRK2 appeared to play a negative role in ERK1/2 activation. Finally, we show that arrestin association with CXCR4 is primarily driven by the phosphorylation of far C-terminal residues on the receptor. These studies reveal that site-specific phosphorylation of CXCR4 is dynamically regulated by multiple kinases resulting in both positive and negative modulation of CXCR4 signaling.     

Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration,activation, and regulation

IL-8 (or CXCL8) activates the receptors CXCR1 (IL-8RA) and CXCR2 (IL-8RB) to induce chemotaxis in leukocytes, but only CXCR1 mediates cytotoxic and cross-regulatory signals. This may be due to the rapid internalization of CXCR2. To investigate the roles of the intracellular domains in receptor regulation, wild-type, chimeric, phosphorylation-deficient, and cytoplasmic tail (C-tail) deletion mutants of both receptors were expressed in RBL-2H3 cells and studied for cellular activation, receptor phosphorylation, desensitization, and internalization. All but one chimeric receptor bound IL-8 and mediated signal transduction, chemotaxis, and exocytosis. Upon IL-8 activation, the chimeric receptors underwent receptor phosphorylation and desensitization. One was resistant to internalization, yet it mediated normal levels of beta-arrestin 2 (beta arr-2) translocation. The lack of internalization by this receptor may be due to its reduced association with beta arr-2 and the adaptor protein-2 beta. The C-tail-deleted and phosphorylation-deficient receptors were resistant to receptor phosphorylation, desensitization, arrestin translocation, and internalization. They also mediated greater phosphoinositide hydrolysis and exocytosis and sustained Ca(2+) mobilization, but diminished chemotaxis. These data indicate that phosphorylation of the C-tails of CXCR1 and CXCR2 are required for arrestin translocation and internalization, but are not sufficient to explain the rapid internalization of CXCR2 relative to CXCR1. The data also show that receptor internalization is not required for chemotaxis. The lack of receptor phosphorylation was correlated with greater signal transduction but diminished chemotaxis, indicating that second messenger production, not receptor internalization, negatively regulates chemotaxis.     

Differential regulation of CXCR4-mediated T-cell chemotaxis and mitogen-activated protein kinase activation by the membrane tyrosine phosphatase,CD45

The chemokine receptor CXCR4 and its cognate ligand, stromal cell-derived factor-1alpha (CXCL12), regulate lymphocyte trafficking and play an important role in host immune surveillance. However, the molecular mechanisms involved in CXCL12-induced and CXCR4-mediated chemotaxis of T-lymphocytes are not completely elucidated. In the present study, we examined the role of the membrane tyrosine phosphatase CD45, which regulates antigen receptor signaling in CXCR4-mediated chemotaxis and mitogen-activated protein kinase (MAPK) activation in T-cells. We observed a significant reduction in CXCL12-induced chemotaxis in the CD45-negative Jurkat cell line (J45.01) as compared with the CD45-positive control (JE6.1) cells. Expression of a chimeric protein containing the intracellular phosphatase domain of CD45 was able to partially restore CXCL12-induced chemotaxis in the J45.01 cells. However, reconstitution of CD45 into the J45.01 cells restored the CXCL12-induced chemotaxis to about 90%. CD45 had no significant effect on CXCL12 or human immunodeficiency virus gp120-induced internalization of the CXCR4 receptor. Furthermore, J45.01 cells showed a slight enhancement in CXCL12-induced MAP kinase activity as compared with the JE6.1 cells. We also observed that CXCL12 treatment enhanced the tyrosine phosphorylation of CD45 and induced its association with the CXCR4 receptor. Pretreatment of T-cells with the lipid raft inhibitor, methyl-beta-cyclodextrin, blocked the association between CXCR4 and CD45 and markedly abolished CXCL12-induced chemotaxis. Comparisons of signaling pathways induced by CXCL12 in JE6.1 and J45.01 cells revealed that CD45 might moderately regulate the tyrosine phosphorylation of the focal adhesion components the related adhesion focal tyrosine kinase/Pyk2, focal adhesion kinase, p130Cas, and paxillin. CD45 has also been shown to regulate CXCR4-mediated activation and phosphorylation of T-cell receptor downstream effectors Lck, ZAP-70, and SLP-76. Our results show that CD45 differentially regulates CXCR4-mediated chemotactic activity and MAPK activation by modulating the activities of focal adhesion components and the downstream effectors of the T-cell receptor.     

Altered CXCR2 signaling in beta-arrestin-2-deficient mouse models

CXCR2 is a G-protein-coupled receptor (GPCR) that binds the CXC chemokines, CXCL1-3 and CXCL5-8, and induces intracellular signals associated with chemotaxis. Many adaptor proteins are actively involved in the sequestration, internalization, and trafficking of CXCR2 and transduction of agonist-induced intracellular signaling. We have previously shown that adaptor protein beta-arrestin-2 (betaarr2) plays a crucial role in transducing signals mediated through CXCR2. To further investigate the role of betaarr2 on CXCR2-mediated signaling during acute inflammation, zymosan-induced neutrophils were isolated from peritoneal cavities of betaarr2-deficient (betaarr2(-/-)) and their wild-type (betaarr2(+/+)) littermate mice, and neutrophil CXCR2 signaling activities were determined by measurement of Ca(2+) mobilization, receptor internalization, GTPase activity, and superoxide anion production. The results showed that the deletion of betaarr2 resulted in increased Ca(2+) mobilization, superoxide anion production, and GTPase activity in neutrophils, but decreased receptor internalization relative to wild-type mice. Two animal models, the dorsal air pouch model and the excisional wound healing model, were used to further study the in vivo effects of betaarr2 on CXCR2-mediated neutrophil chemotaxis and on cutaneous wound healing. Surprisingly, the recruitment of neutrophils was increased in response to CXCL1 in the air pouch model and in the excisional wound beds of betaarr2(-/-) mice. Wound re-epithelialization was also significantly faster in betaarr2(-/-) mice than in betaarr2(+/+) mice. Taken together, the data indicate that betaarr2 is a negative regulator for CXCR2 in vivo signaling.     

Cross-desensitization among CXCR1, CXCR2, and CCR5: role of protein kinase C-epsilon

The IL-8 (or CXCL8) chemokine receptors, CXCR1 and CXCR2, activate protein kinase C (PKC) to mediate leukocyte functions. To investigate the roles of different PKC isoforms in CXCL8 receptor activation and regulation, human mononuclear phagocytes were treated with CXCL8 or CXCL1 (melanoma growth-stimulating activity), which is specific for CXCR2. Plasma membrane association was used as a measure of PKC activation. Both receptors induced time-dependent association of PKCalpha, -beta1, and -beta2 to the membrane, but only CXCR1 activated PKCepsilon. CXCL8 also failed to activate PKCepsilon in RBL-2H3 cells stably expressing CXCR2. DeltaCXCR2, a cytoplasmic tail deletion mutant of CXCR2 that is resistant to internalization, activated PKCepsilon as well as CXCR1. Expression of the PKCepsilon inhibitor peptide epsilonV1 in RBL-2H3 cells blocked PKCepsilon translocation and inhibited receptor-mediated exocytosis, but not phosphoinositide hydrolysis or peak intracellular Ca(2+) mobilization. epsilonV1 also inhibited CXCR1-, CCR5-, and DeltaCXCR2-mediated cross-regulatory signals for GTPase activity, Ca(2+) mobilization, and internalization. Peritoneal macrophages from PKCepsilon-deficient mice (PKCepsilon(-/-)) also showed decreased CCR5-mediated cross-desensitization of G protein activation and Ca(2+) mobilization. Taken together, the results indicate that CXCR1 and CCR5 activate PKCepsilon to mediate cross-inhibitory signals. Inhibition or deletion of PKCepsilon decreases receptor-induced exocytosis and cross-regulatory signals, but not phosphoinositide hydrolysis or peak intracellular Ca(2+) mobilization, suggesting that cross-regulation is a Ca(2+)-independent process. Because DeltaCXCR2, but not CXCR2, activates PKCepsilon and cross-desensitizes CCR5, the data further suggest that signal duration leading to activation of novel PKC may modulate receptor-mediated cross-inhibitory signals.     

G protein-coupled receptor kinase 5 phosphorylation of hip regulates internalization of the chemokine receptor CXCR4

Regulation of the magnitude, duration, and localization of G protein-coupled receptor (GPCR) signaling responses is controlled by desensitization, internalization, and downregulation of the activated receptor. Desensitization is initiated by the phosphorylation of the activated receptor by GPCR kinases (GRKs) and the binding of the adaptor protein arrestin. In addition to phosphorylating activated GPCRs, GRKs have been shown to phosphorylate a variety of additional substrates. An in vitro screen for novel GRK substrates revealed Hsp70 interacting protein (Hip) as a substrate. GRK5, but not GRK2, bound to and stoichiometrically phosphorylated Hip in vitro. The primary binding domain of GRK5 was mapped to residues 303-319 on Hip, while the major site of phosphorylation was identified to be Ser-346. GRK5 also bound to and phosphorylated Hip on Ser-346 in cells. While Hip was previously implicated in chemokine receptor trafficking, we found that the phosphorylation of Ser-346 was required for proper agonist-induced internalization of the chemokine receptor CXCR4. Taken together, Hip has been identified as a novel substrate of GRK5 in vitro and in cells, and phosphorylation of Hip by GRK5 plays a role in modulating CXCR4 internalization.     

CXCR7/CXCR4 heterodimer constitutively recruits beta-arrestin to enhance cell migration

G protein-coupled receptor hetero-oligomerization is emerging as an important regulator of ligand-dependent transmembrane signaling, but precisely how receptor heteromers affect receptor pharmacology remains largely unknown. In this study, we have attempted to identify the functional significance of the heteromeric complex between CXCR4 and CXCR7 chemokine receptors. We demonstrate that co-expression of CXCR7 with CXCR4 results in constitutive recruitment of β-arrestin to the CXCR4·CXCR7 complex and simultaneous impairment of G(i)-mediated signaling. CXCR7/CXCR4 co-expression also results in potentiation of CXCL12 (SDF-1)-mediated downstream β-arrestin-dependent cell signaling pathways, including ERK1/2, p38 MAPK, and SAPK as judged from the results of experiments using siRNA knockdown to deplete β-arrestin. Interestingly, CXCR7/CXCR4 co-expression enhances cell migration in response to CXCL12 stimulation. Again, inhibition of β-arrestin using either siRNA knockdown or a dominant negative mutant abrogates the enhanced CXCL12-dependent migration of CXCR4/CXCR7-expressing cells. These results show how CXCR7, which cannot signal directly through G protein-linked pathways, can nevertheless affect cellular signaling networks by forming a heteromeric complex with CXCR4. The CXCR4·CXCR7 heterodimer complex recruits β-arrestin, resulting in preferential activation of β-arrestin-linked signaling pathways over canonical G protein pathways. CXCL12-dependent signaling of CXCR4 and its role in cellular physiology, including cancer metastasis, should be evaluated in the context of potential functional hetero-oligomerization with CXCR7.     

CXCL14 is no direct modulator of CXCR4

C-X-C motif chemokine 12/C-X-C chemokine receptor type 4 (CXCL12/CXCR4) signaling is involved in ontogenesis, hematopoiesis, immune function and cancer. Recently, the orphan chemokine CXCL14 was reported to inhibit CXCL12-induced chemotaxis – probably by allosteric modulation of CXCR4. We thus examined the effects of CXCL14 on CXCR4 regulation and function using CXCR4-transfected human embryonic kidney (HEK293) cells and Jurkat T cells. CXCL14 did not affect dose–response profiles of CXCL12-induced CXCR4 phosphorylation, G protein-mediated calcium mobilization, dynamic mass redistribution, kinetics of extracellular signal-regulated kinase 1 (ERK1) and ERK2 phosphorylation or CXCR4 internalization. Hence, essential CXCL12-operated functions of CXCR4 are insensitive to CXCL14, suggesting that interactions of CXCL12 and CXCL14 pathways depend on a yet to be identified CXCL14 receptor.     

Cross-regulation of VPAC(2) receptor desensitization by M(3) receptors via PKC-mediated phosphorylation of RKIP and inhibition of GRK2

In gastrointestinal smooth muscle cells, VPAC(2) receptor desensitization is exclusively mediated by G protein-coupled receptor kinase 2 (GRK2). The present study examined the mechanisms by which acetylcholine (ACh) acting via M(3) receptors regulates GRK2-mediated VPAC(2) receptor desensitization in gastric smooth muscle cells. Vasoactive intestinal peptide induced VPAC(2) receptor phosphorylation, internalization, and desensitization in both freshly dispersed and cultured smooth muscle cells. Costimulation with ACh in the presence of M(2) receptor antagonist (i.e., activation of M(3) receptors) inhibited VPAC(2) receptor phosphorylation, internalization, and desensitization. Inhibition was blocked by the selective protein kinase C (PKC) inhibitor bisindolylmaleimide, suggesting that the inhibition was mediated by PKC, derived from M(3) receptor activation. Similar results were obtained by direct activation of PKC with phorbol myristate acetate. In the presence of the M(2) receptor antagonist, ACh induced phosphorylation of Raf kinase inhibitory protein (RKIP), increased RKIP-GRK2 association, decreased RKIP-Raf-1 association, and stimulated ERK1/2 activity, suggesting that, upon phosphorylation by PKC, RKIP dissociates from its known target Raf to associate with, and block the activity of, GRK2. In muscle cells expressing RKIP(S153A), which lacks the PKC phosphorylation site, RKIP phosphorylation was blocked and the inhibitory effect of ACh on VPAC(2) receptor phosphorylation, internalization, and desensitization and the stimulatory effect on ERK1/2 activation were abolished. This study identified a novel mechanism of cross-regulation of G(s)-coupled receptor phosphorylation and internalization by G(q)-coupled receptors. The mechanism involved phosphorylation of RKIP by PKC, switching RKIP from association with Raf-1 to association with, and inhibition of, GRK2.     

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.     

Cross-regulation of VPAC2 receptor internalization by m2 receptors via c-Src-mediated phosphorylation of GRK2

The aim of the study was to examine the mechanisms by which ACh, acting via m2 receptors, regulates GRK2-mediated VPAC(2) receptor desensitization in gastric smooth muscle cells. VIP induced VPAC(2) receptor phosphorylation and internalization in freshly dispersed smooth muscle cells. Co-stimulation with acetylcholine (ACh), in the presence of m3 receptor antagonist, 4-DAMP, augmented VPAC(2) receptor phosphorylation and internalization. The m2 receptor antagonist methoctramine or the c-Src inhibitor PP2 blocked the effect of ACh, suggesting that the augmentation was mediated by c-Src, derived from m2 receptor activation. ACh induced activation of c-Src and phosphorylation of GRK2 and the effects of ACh were blocked by methoctramine, PP2, or by uncoupling of m2 receptors from G(i3) with pertussis toxin. In conclusion, we identified a novel mechanism of cross-regulation of GRK2-mediated phosphorylation and internalization of G(s)-coupled VPAC(2) receptors by G(i)-coupled m2 receptors via tyrosine phosphorylation of GRK2 and stimulation of GRK2 activity.     

Regulation of the human chemokine receptor CCR1. Cross-regulation by CXCR1 and CXCR2

To investigate the regulation of the CCR1 chemokine receptor, a rat basophilic leukemia (RBL-2H3) cell line was modified to stably express epitope-tagged receptor. These cells responded to RANTES (regulated upon activation normal T expressed and secreted), macrophage inflammatory protein-1alpha, and monocyte chemotactic protein-2 to mediate phospholipase C activation, intracellular Ca(2+) mobilization and exocytosis. Upon activation, CCR1 underwent phosphorylation and desensitization as measured by diminished GTPase stimulation and Ca(2+) mobilization. Alanine substitution of specific serine and threonine residues (S2 and S3) or truncation of the cytoplasmic tail (DeltaCCR1) of CCR1 abolished receptor phosphorylation and desensitization of G protein activation but did not abolish desensitization of Ca(2+) mobilization. S2, S3, and DeltaCCR1 were also resistant to internalization, mediated greater phosphatidylinositol hydrolysis and sustained Ca(2+) mobilization, and were only partially desensitized by RANTES, relative to S1 and CCR1. To study CCR1 cross-regulation, RBL cells co-expressing CCR1 and receptors for interleukin-8 (CXCR1, CXCR2, or a phosphorylation-deficient mutant of CXCR2, 331T) were produced. Interleukin-8 stimulation of CXCR1 or CXCR2 cross-phosphorylated CCR1 and cross-desensitized its ability to stimulate GTPase activity and Ca(2+) mobilization. Interestingly, CCR1 cross-phosphorylated and cross-desensitized CXCR2, but not CXCR1. Ca(2+) mobilization by S3 and DeltaCCR1 were also cross-desensitized by CXCR1 and CXCR2 despite lack of receptor phosphorylation. In contrast to wild type CCR1, S3 and DeltaCCR1, which produced sustained signals, cross-phosphorylated and cross-desensitized responses to CXCR1 as well as CXCR2. Taken together, these results indicate that CCR1-mediated responses are regulated at several steps in the signaling pathway, by receptor phosphorylation at the level of receptor/G protein coupling and by an unknown mechanism at the level of phospholipase C activation. Moreover selective cross-regulation among chemokine receptors is, in part, a consequence of the strength of signaling (i.e. greater phosphatidylinositol hydrolysis and sustained Ca(2+) mobilization) which is inversely correlated with the receptor's susceptibility to phosphorylation. Since many chemokines activate multiple chemokine receptors, selective cross-regulation among such receptors may play a role in their immunomodulation.     

A phosphorylation cluster of five serine and threonine residues in the C-terminus of the follicle-stimulating hormone receptor is important for desensitization but not for beta-arrestin-mediated ERK activation

Classically, the FSH receptor (FSH-R) mediates its effects through coupling to guanine nucleotide-binding protein alpha S subunit (Galpha(s)) and activation of the cAMP/protein kinase A (PKA) signaling pathway. beta-Arrestins are rapidly recruited to the FSH-activated receptor and play key roles in its desensitization and internalization. Here, we show that the FSH-R expressed in HEK 293 cells activated ERK by two temporally distinct pathways dependent, respectively, on Galpha(s)/PKA and beta-arrestins. Galpha(s)/PKA-dependent ERK activation was rapid, transient, and blocked by H89 (a PKA inhibitor), but it was insensitive to small interfering RNA-mediated depletion of beta-arrestins. beta-Arrestin-dependent ERK activation was slower but more sustained and was insensitive to H89. We identified five Ser/Thr residues in the C terminus of the receptor (638-644) as a major phosphorylation site. Mutation of these residues into Ala (5A FSH-R) significantly reduced the stability of FSH-induced beta-arrestin 1 and 2 interaction when compared with the wild-type receptor. As expected, the 5A FSH-R-mediated cAMP accumulation was enhanced, and its internalization was reduced. In striking contrast, the ability of the 5A FSH-R to activate ERK via the beta-arrestin-dependent pathway was increased. G protein-coupled receptor kinase 5 (GRK5) and GRK6 were required for beta-arrestin-dependent ERK activation by both the wild-type and 5A FSH-R. By contrast, GRK2 depletion enhanced ERK activation by the wild-type FSH-R but not by the 5A FSH-R. In conclusion, we demonstrate the existence of a beta-arrestin-dependent, GRK-regulated mechanism for ERK activation by the FSH-R. A phosphorylation cluster in the C terminus of the FSH-R, identified as a site of beta-arrestin recruitment, positively regulated both desensitization and internalization but negatively regulated beta-arrestin-dependent ERK activation.     

Physiological changes in GRK2 regulate CCL2-induced signaling to ERK1/2 and Akt but not to MEK1/2 and calcium

G protein-coupled receptor (GPCR) kinase 2 (GRK2) regulates G protein-coupled receptor signaling via agonist-induced receptor phosphorylation and desensitization. GRK2 can also modulate cellular activation by interacting with downstream signaling molecules. The intracellular GRK2 level changes during inflammatory conditions. We investigated how IL-1β-induced changes in endogenous GRK2 expression influence chemokine receptor signaling in primary astrocytes. Culturing astrocytes with IL-1β for 24 h induced a 2–3-fold increase in GRK2 and decreased C–C chemokine ligand 2 (CCL2)-induced ERK1/2 activation. Conversely, the 45% decrease in GRK2 expression in astrocytes from GRK2+/− animals resulted in a more pronounced CCL2-induced ERK1/2 phosphorylation. Increased GRK2 inhibited CCL2-induced Akt phosphorylation at Thr308 and Ser473 as well as pPDK-1 translocation. In contrast, altered GRK2 levels did not change the CCL2-induced increase in intracellular calcium or MEK1/2 phosphorylation. These data suggest that altered GRK2 expression modulates chemokine signaling downstream of the receptor. We found that GRK2 kinase activity was not required to decrease chemokine-induced ERK1/2 phosphorylation, whereas regulation of CCL2-induced Akt phosphorylation did require an active GRK2 kinase domain. Collectively, these data suggest that changes in endogenous GRK2 expression in primary astrocytes regulate chemokine receptor signaling to ERK1/2 and to PDK-1-Akt downstream of receptor coupling via kinase-dependent and kinase-independent mechanisms, respectively.     

Role of the G protein-coupled receptor kinase site serine cluster in beta2-adrenergic receptor internalization, desensitization, and beta-arrestin translocation

There is considerable evidence for the role of carboxyl-terminal serines 355, 356, and 364 in G protein-coupled receptor kinase (GRK)-mediated phosphorylation and desensitization of beta(2)-adrenergic receptors (beta(2)ARs). In this study we used receptors in which these serines were changed to alanines (SA3) or to aspartic acids (SD3) to determine the role of these sites in beta-arrestin-dependent beta(2)AR internalization and desensitization. Coupling efficiencies for epinephrine activation of adenylyl cyclase were similar in wild-type and mutant receptors, demonstrating that the SD3 mutant did not drive constitutive GRK desensitization. Treatment of wild-type and mutant receptors with 0.3 nm isoproterenol for 5 min induced approximately 2-fold increases in the EC(50) for agonist activation of adenylyl cyclase, consistent with protein kinase A (PKA) site-mediated desensitization. When exposed to 1 mum isoproterenol to trigger GRK site-mediated desensitization, only wild-type receptors showed significant further desensitization. Using a phospho site-specific antibody, we determined that there is no requirement for these GRK sites in PKA-mediated phosphorylation at high agonist concentration. The rates of agonist-induced internalization of the SD3 and SA3 mutants were 44 and 13%, respectively, relative to that of wild-type receptors, but the SD3 mutant recruited enhanced green fluorescent protein (EGFP)-beta-arrestin 2 to the plasma membrane, whereas the SA3 mutant did not. EGFP-beta-Arrestin2 overexpression triggered a significant increase in the extent of SD3 mutant desensitization but had no effect on the desensitization of wild-type receptors or the SA3 mutant. Expression of a phosphorylation-independent beta-arrestin 1 mutant (R169E) significantly rescued the internalization defect of the SA3 mutant but inhibited the phosphorylation of serines 355 and 356 in wild-type receptors. Our data demonstrate that (i) the lack of GRK sites does not impair PKA site phosphorylation, (ii) the SD3 mutation inhibits GRK-mediated desensitization although it supports some agonist-induced beta-arrestin binding and receptor internalization, and (iii) serines 355, 356, and 364 play a pivotal role in the GRK-mediated desensitization, beta-arrestin binding, and internalization of beta(2)ARs.     

Protein kinase A and G protein-coupled receptor kinase phosphorylation mediates beta-1 adrenergic receptor endocytosis through different pathways

Agonist-induced phosphorylation of beta-adrenergic receptors (beta ARs) by G protein-coupled receptor kinases (GRKs) results in their desensitization followed by internalization. Whether protein kinase A (PKA)-mediated phosphorylation of beta ARs, particularly the beta 1AR subtype, can also trigger internalization is currently not known. To test this, we cloned the mouse wild type beta 1AR (WT beta 1AR) and created 3 mutants lacking, respectively: the putative PKA phosphorylation sites (PKA-beta 1AR), the putative GRK phosphorylation sites (GRK-beta 1AR), and both sets of phosphorylation sites (PKA-/GRK-beta 1AR). Following agonist stimulation, both PKA-beta 1AR and GRK-beta 1AR mutants showed comparable increases in phosphorylation and desensitization. Saturating concentrations of agonist induced only 50% internalization of either mutant compared with wild type, suggesting that both PKA and GRK phosphorylation of the receptor contributed to receptor sequestration in an additive manner. Moreover, in contrast to the WT beta 1AR and PKA-beta 1AR, sequestration of the GRK-beta 1AR and PKA-/GRK-beta 1AR was independent of beta-arrestin recruitment. Importantly, clathrin inhibitors abolished agonist-dependent internalization for both the WT beta 1AR and PKA-beta 1AR, whereas caveolae inhibitors prevented internalization only of the GRK-beta 1AR mutant. Taken together, these data demonstrate that: 1) PKA-mediated phosphorylation can trigger agonist-induced internalization of the beta 1AR and 2) the pathway selected for beta 1AR internalization is primarily determined by the kinase that phosphorylates the receptor, i.e. PKA-mediated phosphorylation directs internalization via a caveolae pathway, whereas GRK-mediated phosphorylation directs it through clathrin-coated pits.     

Regulation of C3a receptor signaling in human mast cells by G protein coupled receptor kinases

Background

The complement component C3a activates human mast cells via its cell surface G protein coupled receptor (GPCR) C3aR. For most GPCRs, agonist-induced receptor phosphorylation leads to receptor desensitization, internalization as well as activation of downstream signaling pathways such as ERK1/2 phosphorylation. Previous studies in transfected COS cells overexpressing G protein coupled receptor kinases (GRKs) demonstrated that GRK2, GRK3, GRK5 and GRK6 participate in agonist-induced C3aR phosphorylation. However, the roles of these GRKs on the regulation of C3aR signaling and mediator release in human mast cells remain unknown.

Methodology/Principal Findings

We utilized lentivirus short hairpin (sh)RNA to stably knockdown the expression of GRK2, GRK3, GRK5 and GRK6 in human mast cell lines, HMC-1 and LAD2, that endogenously express C3aR. Silencing GRK2 or GRK3 expression caused a more sustained Ca²⁺ mobilization, attenuated C3aR desensitization, and enhanced degranulation as well as ERK1/2 phosphorylation when compared to shRNA control cells. By contrast, GRK5 or GRK6 knockdown had no effect on C3aR desensitization, but caused a significant decrease in C3a-induced mast cell degranulation. Interestingly, GRK5 or GRK6 knockdown rendered mast cells more responsive to C3a for ERK1/2 phosphorylation.

Conclusion/Significance

This study demonstrates that GRK2 and GRK3 are involved in C3aR desensitization. Furthermore, it reveals the novel finding that GRK5 and GRK6 promote C3a-induced mast cell degranulation but inhibit ERK1/2 phosphorylation via C3aR desensitization-independent mechanisms. These findings thus reveal a new level of complexity for C3aR regulation by GRKs in human mast cells.     

Chemokine receptor CXCR3 desensitization by IL-16/CD4 signaling is dependent on CCR5 and intact membrane cholesterol

Previous work has shown that IL-16/CD4 induces desensitization of both CCR5- and CXCR4-induced migration, with no apparent effect on CCR2b or CCR3. To investigate the functional relationship between CD4 and other chemokine receptors, we determined the effects of IL-16 interaction with CD4 on CXCR3-induced migration. In this study we demonstrate that IL-16/CD4 induced receptor desensitization of CXCR3 on primary human T cells. IL-16/CD4 stimulation does not result in surface modulation of CXCR3 or changes in CXCL10 binding affinity. This effect does require p56(lck) enzymatic activity and the presence of CCR5, because desensitization is not transmitted in the absence of CCR5. Treatment of human T cells with methyl-beta-cyclodextrin, a cholesterol chelator, prevented the desensitization of CXCR3 via IL-16/CD4, which was restored after reloading of cholesterol, indicating a requirement for intact cholesterol. These studies demonstrate an intimate functional relationship among CD4, CCR5, and CXCR3, in which CCR5 can act as an adaptor molecule for CD4 signaling. This process of regulating Th1 cell chemoattraction may represent a mechanism for orchestrating cell recruitment in Th1-mediated diseases.     

Phosphorylation of the platelet-derived growth factor receptor-beta by G protein-coupled receptor kinase-2 reduces receptor signaling and interaction with the Na(+)/H(+) exchanger regulatory factor

G protein-coupled receptor kinase-2 (GRK2) can phosphorylate and desensitize the platelet-derived growth factor receptor-beta (PDGFRbeta) in heterologous cellular systems. To determine whether GRK2 regulates the PDGFRbeta in physiologic systems, we examined PDGFRbeta signaling in mouse embryonic fibroblasts from GRK2-null and cognate wild type mice. To discern a mechanism by which GRK2-mediated phosphorylation can desensitize the PDGFRbeta, but not the epidermal growth factor receptor (EGFR), we investigated effects of GRK2-mediated phosphorylation on the association of the PDGFRbeta with the Na(+)/H(+) exchanger regulatory factor (NHERF), a protein shown to potentiate dimerization of the PDGFRbeta, but not the EGFR. Physiologic expression of GRK2 diminished (a) phosphoinositide hydrolysis elicited through the PDGFRbeta but not heterotrimeric G proteins; (b) Akt activation evoked by the PDGFRbeta but not the EGFR; and (c) PDGF-induced tyrosyl phosphorylation of the PDGFRbeta itself. PDGFRbeta desensitization by physiologically expressed GRK2 correlated with a 2.5-fold increase in PDGF-promoted PDGFRbeta seryl phosphorylation. In 293 cells, GRK2 overexpression reduced PDGFRbeta/NHERF association by 60%. This effect was reproduced by S1104D mutation of the PDGFRbeta, which also diminished PDGFRbeta activation and signaling (like the S1104A mutation) to an extent equivalent to that achieved by GRK2-mediated PDGFRbeta phosphorylation. GRK2 overexpression desensitized only the wild type but not the S1104A PDGFRbeta. We conclude that GRK2-mediated PDGFRbeta seryl phosphorylation plays an important role in desensitizing the PDGFRbeta in physiologic systems. Furthermore, this desensitization appears to involve GRK2-mediated phosphorylation of PDGFRbeta Ser(1104), with consequent dissociation of the PDGFRbeta from NHERF.     

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.