Different mechanisms regulate lysophosphatidic acid (LPA)-dependent versus phorbol ester-dependent internalization of the LPA1 receptor

Lysophosphatidic acid (LPA) stimulates cells by activation of five G-protein-coupled receptors, termed LPA 1-5. The LPA 1 receptor is the most widely expressed and is a major regulator of cell migration. In this study, we show that phorbol ester (PMA)-induced internalization of the LPA(1) receptor requires clathrin AP-2 complexes, protein kinase C, and a distal dileucine motif (amino acids 352 and 353) in the cytoplasmic tail but not beta-arrestin. Agonist-dependent internalization of LPA 1, however, requires a cluster of serine residues (amino acids 341-347) located proximal to the dileucine motif, beta-arrestin, and to a lesser extent clathrin AP-2. The serine cluster of LPA 1 is required for beta-arrestin2-GFP translocation to the plasma membrane and signal desensitization. In contrast, the dileucine motif (IL) is required for both basal and PMA-induced internalization. Evidence for the beta-arrestin independence of PMA-induced internalization of LPA 1 comes from the observations that beta-arrestin2-GFP is not recruited to the plasma membrane upon PMA treatment and that LPA 1 is readily internalized in beta-arrestin1/2 knock-out mouse embryonic fibroblasts. These results indicate that distinct molecular mechanisms regulate agonist-dependent and PMA-dependent internalization of the LPA 1 receptor.     

Phorbol Esters and SDF-1 Induce Rapid Endocytosis and Down Modulation of the Chemokine Receptor CXCR4

The chemokine receptor CXCR4 is required, together with CD4, for entry by some isolates of HIV-1, particularly those that emerge late in infection. The use of CXCR4 by these viruses likely has profound effects on viral host range and correlates with the evolution of immunodeficiency. Stromal cell-derived factor-1 (SDF-1), the ligand for CXCR4, can inhibit infection by CXCR4-dependent viruses. To understand the mechanism of this inhibition, we used a monoclonal antibody that is specific for CXCR4 to analyze the effects of phorbol esters and SDF-1 on surface expression of CXCR4. On human T cell lines SupT1 and BC7, CXCR4 undergoes slow constitutive internalization (1.0% of the cell surface pool/min). Addition of phorbol esters increased this endocytosis rate >6-fold and reduced cell surface CXCR4 expression by 60 to 90% over 120 min. CXCR4 was internalized through coated pits and coated vesicles and subsequently localized in endosomal compartments from where it could recycle to the cell surface after removal of the phorbol ester. SDF-1 also induced the rapid down modulation (half time ~5 min) of CXCR4. Using mink lung epithelial cells expressing CXCR4 and a COOH-terminal deletion mutant of CXCR4, we found that an intact cytoplasmic COOH-terminal domain was required for both PMA and ligand-induced CXCR4 endocytosis. However, experiments using inhibitors of protein kinase C indicated that SDF-1 and phorbol esters trigger down modulation through different cellular mechanisms.

SDF-1 inhibited HIV-1 infection of mink cells expressing CD4 and CXCR4. The inhibition of infection was less efficient for CXCR4 lacking the COOH-terminal domain, suggesting at least in part that SDF-1 inhibition of virus infection was mediated through ligand-induced internalization of CXCR4. Significantly, ligand induced internalization of CXCR4 but not CD4, suggesting that CXCR4 and CD4 do not normally physically interact on the cell surface. Together these studies indicate that endocytosis can regulate the cell-surface expression of CXCR4 and that SDF-1–mediated down regulation of cell-surface coreceptor expression contributes to chemokine-mediated inhibition of HIV infection.

     

Loss of chemokine SDF-1alpha-mediated CXCR4 signalling and receptor internalization in human hepatoma cell line HepG2

Expression of the chemokine stromal cell-derived factor-1alpha (SDF-1alpha) is absent from many carcinomas, including hepatomas. We note an early signalling defect in the hepatocellular carcinoma (HCC) cell line HepG2 that expresses the CXCR4 receptor and binds biotin-labelled SDF, but fails to stimulate downstream signalling events after engagement with SDF. In HepG2, the SDF/CXCR4 interaction did not result in calcium influx, phosphorylation and internalization of CXCR4, nor in a rapid phosphorylation of p44/42 MAP kinase. There were no CXCR4 mutations in the second chemokine binding loop or C terminal phosphorylation and internalization domains. The downstream signalling machinery in HepG2 appears to be intact since transfection of wild-type CXCR4 restored functional responsiveness. We conclude that HepG2 is unresponsive to SDF stimulation because of a defect located after receptor binding but before the activation of the signalling cascade. A hypothetical blocking molecule could hinder receptor internalization or CXCR4 signalling.     

Agonist-promoted ubiquitination of the G protein-coupled receptor CXCR4 mediates lysosomal sorting

Ligand-induced trafficking plays an important role in the physiologic regulation of many G protein-coupled receptors (GPCRs). Although numerous GPCRs are sorted to a degradative pathway upon prolonged stimulation, the molecular events leading to degradation are poorly understood. Here we report that the human immunodeficiency virus co-receptor CXCR4 undergoes rapid agonist-promoted degradation by a process involving endocytosis via clathrin-coated pits and subsequent sorting to lysosomes. Studies analyzing the sorting of various CXCR4 mutants revealed the presence of a degradation motif (SSLKILSKGK) in the carboxyl terminus of CXCR4. The first two serines as well as the dileucine motif were critical for agonist-induced endocytosis, whereas all three serines but not the dileucine were important in mediating degradation. Mutation of the three lysine residues had no effect on CXCR4 endocytosis yet completely inhibited receptor degradation. Because lysine residues represent potential sites of ubiquitination, we also examined the ubiquitination of CXCR4. Interestingly, CXCR4 was shown to undergo rapid agonist-promoted ubiquitination that was attenuated by mutation of the lysine residues within the degradation motif. These studies implicate a specific role for ubiquitination in sorting endocytosed GPCRs to lysosomes.     

Chemokine receptor inhibition by AMD3100 is strictly confined to CXCR4

This study was undertaken to demonstrate the unique specificity of the chemokine receptor CXCR4 antagonist AMD3100. Calcium flux assays with selected chemokine/cell combinations, affording distinct chemokine receptor specificities, revealed no interaction of AMD3100 with any of the chemokine receptors CXCR1 through CXCR3, or CCR1 through CCR9. In contrast, AMD3100 potently inhibited CXCR4-mediated calcium signaling and chemotaxis in a concentration-dependent manner in different cell types. Also, AMD3100 inhibited stromal cell-derived factor (SDF)-1-induced endocytosis of CXCR4, but did not affect phorbol ester-induced receptor internalization. Importantly, AMD3100 by itself was unable to elicit intracellular calcium fluxes, to induce chemotaxis, or to trigger CXCR4 internalization, indicating that the compound does not act as a CXCR4 agonist. Specific small-molecule CXCR4 antagonists such as AMD3100 may play an important role in the treatment of human immunodeficiency virus infections and many other pathological processes that are dependent on SDF-1/CXCR4 interactions (e.g. rheumatoid arthritis, atherosclerosis, asthma and breast cancer metastasis).     

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.     

Mechanisms of homomeric alpha1 glycine receptor endocytosis

Little is known regarding the mechanism(s) by which glycine receptors are endocytosed. Here we examined the endocytosis of homomeric alpha1 glycine receptors expressed in HEK 293 cells using immunofluorescence/confocal microscopy and whole-cell patch-clamp recordings. Our studies demonstrate that constitutive endocytosis of glycine receptors is blocked by the dominant negative dynamin construct K44A and that intracellular dialysis with peptide P4, a dynamin/amphiphysin-disrupting peptide, increased whole-cell glycine-gated chloride currents. To examine whether receptor endocytosis could be regulated by PKC, experiments with the PKC activator PMA (phorbol 12-myristate 13-acetate) were performed. PMA, but not its inactive analogue PMM (phorbol 12-monomyristate), stimulated receptor endocytosis and inhibited glycine-gated chloride currents. Similar to constitutive endocytosis, PKC-stimulated endocytosis was blocked by dynamin K44A. Mutation of a putative AP2 adaptin dileucine motif (L314A, L315A) present in the receptor cytoplasmic loop blocked PMA-stimulated receptor endocytosis and also prevented PMA inhibition of glycine receptor currents. In patch-clamp experiments, intracellular dialysis of a 12-amino acid peptide corresponding to the region of the receptor containing the dileucine motif prevented PKC modulation of wild-type glycine receptors. Unlike PKC modulation of the receptor, constitutive endocytosis was not affected by mutation of this dileucine motif. These results demonstrate that PKC activation stimulates glycine receptor endocytosis, that both constitutive endocytosis and PKC-stimulated endocytosis are dynamin-dependent, and that PKC-stimulated endocytosis, but not constitutive endocytosis, occurs via the dileucine motif (L314A, L315A) within the cytoplasmic loop of the receptor.     

Stromal cell-derived factor-1alpha modulation of the excitability of rat substantia nigra dopaminergic neurones: presynaptic mechanisms

In rat substantia nigra (SN), Chemokine (CXC motif) receptor 4 (CXCR4) for the chemokine stromal cell-derived factor (SDF)-1alpha is expressed on dopaminergic (DA) neurones, but also on non-DA cells, suggesting presynaptic actions. Using whole-cell patch-clamp recordings in DA neurones of rat SN slices at a holding potential of -60 mV, we showed here that SDF-1alpha exerts multiple presynaptic effects. First, SDF-1alpha (10 nm) induced an increase in the frequency of spontaneous and miniature GABA(A) postsynaptic currents by presynaptic mechanisms, consistent with the presence of CXCR4 on GABAergic neurones of the SN, as revealed by immunocytochemistry. Second, SDF-1alpha (0.1-1 nm) induced a glutamatergic inward current resistant to tetrodotoxin (TTX), most probably the result of glutamate release from non-neuronal cells. This inward current was not blocked by the CXCR4 antagonist AMD 3100 (1 microm), consistent with the lack of CXCR4 on astrocytes as shown by immunocytochemistry under basal conditions. Finally, SDF-1alpha (10 nm) induced, via CXCR4, an outward G protein-activated inward rectifier (GIRK) current, which was TTX sensitive and prevented by application of the GABA(B) antagonist CGP55845A, suggesting GABA spillover on to GABA(B) receptors. Our results show that SDF-1alpha induces, via presynaptic mechanisms, alterations in the excitability of DA neurones as confirmed by current-clamp experiments.     

Protein kinase C activation stimulates the phosphorylation and internalization of the sst2A somatostatin receptor

The sst2A receptor is expressed in the endocrine, gastrointestinal, and neuronal systems as well as in many hormone-sensitive tumors. This receptor is rapidly internalized and phosphorylated in growth hormone-R2 pituitary cells following somatostatin binding (Hipkin, R. W., Friedman, J., Clark, R. B., Eppler, C. M., and Schonbrunn, A. (1997) J. Biol. Chem. 272, 13869-13876). The protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA), also stimulates sst2A phosphorylation. Here we examine the mechanisms and consequences of PMA and agonist-induced sst2A phosphorylation. Like somatostatin, both PMA and bombesin increased sst2A receptor phosphorylation within 2 min. The PKC inhibitor GF109203X blocked PMA- and bombesin- stimulated sst2A phosphorylation, whereas stimulation by the somatostatin analog SMS 201-995 was unaffected. Agonist and PMA each stimulated phosphorylation in two receptor domains, the third intracellular loop and the C-terminal tail. Functionally, PMA dramatically increased the internalization of the sst2A receptor-ligand complex. This PMA stimulation was blocked by GF109203X, whereas basal internalization was unaffected. However, neither basal nor PMA-stimulated internalization was altered by pertussis toxin, whereas both were blocked by hypertonic sucrose. Therefore PKC activation and agonist binding stimulate sst2A phosphorylation by distinct mechanisms, and PKC potentiates internalization of the sst2A receptor via clathrin-coated pits. Thus, hormonal stimulation of PKC-coupled receptors may provide a mechanism for regulating the inhibitory actions of somatostatin in target tissue.     

CXCR4 function requires membrane cholesterol: implications for HIV infection

HIV requires cholesterol and lipid rafts on target cell membranes for infection. To elucidate a possible mechanism, we determined that cholesterol extraction by hydroxypropyl-beta-cyclodextrin (BCD) inhibits stromal cell-derived factor 1alpha (SDF-1alpha) binding to CXCR4 on T cell lines and PBMCs. Intracellular calcium responses to SDF-1alpha, as well as receptor internalization, were impaired in treated T cells. Loss in ligand binding is likely due to conformational changes in CXCR4 and not increased sensitivity to internalization. SDF-1alpha binding and calcium responses were effectively restored by reloading cholesterol. Immunofluorescence microscopy revealed that SDF-1alpha binding occurred in lipid raft microdomains that contained GM1. CXCR4 surface expression, on the other hand, only partially colocalized with GM1. HIV-1(IIIB) infection assays confirmed the functional loss of CXCR4 in the cell lines tested, Sup-T1 and CEM-NKR-CCR5. These data suggest that cholesterol is essential for CXCR4 conformation and function and that lipid rafts may play a regulatory role in SDF-1alpha signaling.     

The chemokine receptors CXCR1 and CXCR2 couple to distinct g protein-coupled receptor kinases to mediate and regulate leukocyte functions

The chemokine receptors, CXCR1 and CXCR2, couple to Gαi to induce leukocyte recruitment and activation at sites of inflammation. Upon activation by CXCL8, these receptors become phosphorylated, desensitized, and internalized. In this study, we investigated the role of different G protein-coupled receptor kinases (GRKs) in CXCR1- and CXCR2-mediated cellular functions. To that end, short hairpin RNA was used to inhibit GRK2, 3, 5, and 6 in RBL-2H3 cells stably expressing CXCR1 or CXCR2, and CXCL8-mediated receptor activation and regulation were assessed. Inhibition of GRK2 and GRK6 increased CXCR1 and CXCR2 resistance to phosphorylation, desensitization, and internalization, respectively, and enhanced CXCL8-induced phosphoinositide hydrolysis and exocytosis in vitro. GRK2 depletion diminished CXCR1-induced ERK1/2 phosphorylation but had no effect on CXCR2-induced ERK1/2 phosphorylation. GRK6 depletion had no significant effect on CXCR1 function. However, peritoneal neutrophils from mice deficient in GRK6 (GRK6(-/-)) displayed an increase in CXCR2-mediated G protein activation but in vitro exhibited a decrease in chemotaxis, receptor desensitization, and internalization relative to wild-type (GRK6(+/+)) cells. In contrast, neutrophil recruitment in vivo in GRK6(-/-) mice was increased in response to delivery of CXCL1 through the air pouch model. In a wound-closure assay, GRK6(-/-) mice showed enhanced myeloperoxidase activity, suggesting enhanced neutrophil recruitment, and faster wound closure compared with GRK6(+/+) animals. Taken together, the results indicate that CXCR1 and CXCR2 couple to distinct GRK isoforms to mediate and regulate inflammatory responses. CXCR1 predominantly couples to GRK2, whereas CXCR2 interacts with GRK6 to negatively regulate receptor sensitization and trafficking, thus affecting cell signaling and angiogenesis.     

Control of receptor internalization, signaling level, and precise arrival at the target in guided cell migration

Activation of the chemokine receptor CXCR4 by SDF1 controls a variety of biological processes in development, immune response, and disease [1-5]. The carboxyl-terminal region of CXCR4 is subject to phosphorylation that allows binding of regulatory proteins [5]; this results in downregulation of CXCR4 signaling and receptor internalization [6]. Notably, truncations of this part of CXCR4 have been implicated in WHIM syndrome, a dominantly inherited immunodeficiency disorder [7, 8]. Despite its importance in receptor signaling and the clinical relevance of its regulation, the precise function of regulating signaling level and internalization in controlling cell behavior is not known. Whereas a number of in vitro studies suggested that the carboxyl terminus of CXCR4 positively regulates chemotaxis (e.g., [9]), others reached the opposite conclusion [8, 10, 11]. These conflicting results highlight the importance of investigating this process under physiological conditions in the live animal. In this study, we demonstrate the significance of internalization and of controlling receptor signaling level for SDF-1-guided migration. We found that whereas internalization and the control over signaling intensity are dispensable for cell motility and directional sensing, they are essential for fine-tuning of migration in vivo, allowing precise arrival of zebrafish PGCs at their target, the region where the gonad develops.     

Structural determinants of unexpected agonist activity in a retro-peptide analogue of the SDF-1alpha N-terminus

We have synthesised two retro-peptide analogues of the stromal cell derived growth factor 1 (SDF-1alpha) segment known to be critical for CXCR4 receptor binding, corresponding to the sequences HSEFFRCPCRFFESH and HSEFFRGGGRFFESH. We have assayed the ability of these peptides to activate extracellular signal-regulated kinase 1/2 phosphorylation in cells over expressing the SDF-1alpha receptor, finding that the first variant was able to serve as an agonist of CXCR4, whereas the second one was inactive. Finally, by comparing representative solution structures of the two peptides, we have found that the biological response of HSEFFRCPCRFFESH may be ascribed to a beta-beta-type turn motif centred on Phe(4)-Phe(5).     

Inhibition of tyrosine kinase activation blocks the down-regulation of CXC chemokine receptor 4 by HIV-1 gp120 in CD4+ T cells.

Because the binding of HIV-1 envelope to CD4 initiates a configurational change in glycoprotein 120 (gp120), enabling it to interact with fusion coreceptors, we investigated how this process interferes with the expression and function of CXC chemokine receptor 4 (CXCR4) in CD4+ T lymphocytes. A recombinant gp120 (MN), after preincubation with CD4+ T lymphocytes, significantly inhibited the binding and chemotaxis of the cells in response to the CXCR4 ligand stromal cell-derived factor-1alpha (SDF-1alpha), accompanied by a markedly reduced surface expression of CXCR4. gp120, but not SDF-1alpha, induced rapid tyrosine phosphorylation of src-like kinase p56lck in CD4+ T cells, whereas both gp120 and SDF-1alpha caused phosphorylation of the CXCR4. The tyrosine kinase inhibitor herbimycin A abolished the phosphorylation of p56lck and CXCR4 induced by gp120 in association with maintenance of normal expression of cell surface CXCR4 and a migratory response to SDF-1alpha. Thus, a CD4-associated signaling molecule(s) including p56lck is activated by gp120 and is required for the down-regulation of CXCR4.     

Glial and neuronal cells express functional chemokine receptor CXCR4 and its natural ligand stromal cell-derived factor 1

Chemokines are a family of proteins that chemoattract and activate cells by interacting with specific receptors on the surface of their targets. The chemokine stromal cell-derived factor 1, (SDF1), binds to the seven-transmembrane G protein-coupled CXCR4 receptor and acts to modulate cell migration, differentiation, and proliferation. CXCR4 and SDF1 are reported to be expressed in various tissues including brain. Here we show that SDF1 and CXCR4 are expressed in cultured cortical type I rat astrocytes, cortical neurons, and cerebellar granule cells. In cortical astrocytes, prolonged treatment with lipopolysaccharide induced an increase of SDF1 expression and a down-regulation of CXCR4, whereas treatment with phorbol esters did not affect SDF1 expression and down-modulated CXCR4 receptor expression. We also demonstrated the ability of human SDF1alpha (hSDF1alpha) to increase the intracellular calcium level in cultured astrocytes and cortical neurons, whereas in the same conditions, cerebellar granule cells did not modify their intracellular calcium concentration. Furthermore, in cortical astrocytes, the simultaneous treatment of hSDF1alpha with the HIV-1 capside glycoprotein gp120 inhibits the cyclic AMP formation induced by forskolin treatment.     

Optimal inhibition of X4 HIV isolates by the CXC chemokine stromal cell-derived factor 1 alpha requires interaction with cell surface heparan sulfate proteoglycans

The chemokine stromal cell-derived factor 1 (SDF-1) is the natural ligand for CXC chemokine receptor 4 (CXCR4). SDF-1 inhibits infection of CD4+ cells by X4 (CXCR4-dependent) human immunodeficiency virus (HIV) strains. We previously showed that SDF-1 alpha interacts specifically with heparin or heparan sulfates (HSs). Herein, we delimited the boundaries of the HS-binding domain located in the first beta-strand of SDF-1 alpha as the critical residues. We also provide evidence that binding to cell surface heparan sulfate proteoglycans (HSPGs) determines the capacity of SDF-1 alpha to prevent the fusogenic activity of HIV-1 X4 isolates in leukocytes. Indeed, SDF-1 alpha mutants lacking the capacity to interact with HSPGs showed a substantially reduced capacity to prevent cell-to-cell fusion mediated by X4 HIV envelope glycoproteins. Moreover, the enzymatic removal of cell surface HS diminishes the HIV-inhibitory capacity of the chemokine to the levels shown by the HS-binding-disabled mutant counterparts. The mechanisms underlying the optimal HIV-inhibitory activity of SDF-1 alpha when attached to HSPGs were investigated. Combining fluorescence resonance energy transfer and laser confocal microscopy, we demonstrate the concomitant binding of SDF-1 alpha to CXCR4 and HSPGs at the cell membrane. Using FRET between a Texas Red-labeled SDF-1 alpha and an enhanced green fluorescent protein-tagged CXCR4, we show that binding of SDF-1 alpha to cell surface HSPGs modifies neither the kinetics of occupancy nor activation in real time of CXCR4 by the chemokine. Moreover, attachment to HSPGs does not modify the potency of the chemokine to promote internalization of CXCR4. Attachment to cellular HSPGs may co-operate in the optimal anti-HIV activity of SDF-1 alpha by increasing the local concentration of the chemokine in the surrounding environment of CXCR4, thus facilitating sustained occupancy and down-regulation of the HIV coreceptor.     

Characterization of sequence determinants within the carboxyl-terminal domain of chemokine receptor CCR5 that regulate signaling and receptor internalization.

The CC chemokine receptor CCR5 mediates chemotaxis of leukocytes and serves as a principal co-receptor for macrophage-tropic human immunodeficiency virus type 1. To identify determinants on the CCR5 carboxyl-terminal domain that regulate receptor signaling and internalization, we generated several CCR5 mutants, which were progressively shortened from the COOH terminus or had carboxyl-terminal serine, cysteine, or leucine residues substituted by alanine and expressed them in RBL-2H3 cells. Using fluorescence resonance energy transfer between beta-arrestin and CCR5 tagged with cyan and yellow variants of green fluorescent protein, we show that high affinity association of the two molecules in living cells requires intact carboxyl-terminal serine phosphorylation sites. Phosphorylation-deficient truncation or Ser/Ala replacement mutants of CCR5 mediated a sustained calcium response and enhanced granular enzyme release in RANTES-stimulated cells. Carboxyl-terminal serine residues are critically involved in CCR5 endocytosis and a dileucine motif, similar to that implicated in the regulation of CXCR2 and CXCR4, contributes to the internalization of CCR5 in a phosphorylation-independent manner. Despite their prominent role in receptor desensitization and internalization, beta-arrestins are dispensable for the CCR5-mediated stimulation of mitogen-activated protein kinase pathways in RBL-2H3 cells. We also show that CCR5 is palmitoylated on carboxyl-terminal cysteine residues. Inhibition of CCR5 palmitoylation by alanine mutagenesis of cysteines or treatment with a palmitate analogue inhibitor profoundly reduces phorbol 12-myristate 13-acetate- and RANTES-induced receptor phosphorylation, homologous desensitization, and internalization. Alanine mutagenesis of serine, cysteine, or leucine residues or the limited carboxyl-terminal truncation of CCR5 did not impair chemokine-stimulated migration of RBL-2H3 cells. Together these results indicate that post-translational modifications of carboxyl-terminal serine and cysteine residues have a significant impact on receptor deactivation and internalization.     

Role of G protein-coupled receptor kinases on the agonist-induced phosphorylation and internalization of the follitropin receptor.

The experiments presented herein were designed to identify members of the G protein-coupled receptor kinase (GRK) family that participate in the agonist-induced phosphorylation and internalization of the rat FSH receptor (rFSHR). Western blots of human kidney 293 cells (the cell line used in transfection experiments) and MSC-1 cells (a cell line derived from Sertoli cells that displays many of the differentiated functions of their normal counterparts) reveal the presence of GRK2 and GRK6 in both cell lines as well as GRK4 in MSC-1 cells. Cotransfection of 293 cells with the rFSHR and GRK2, GRK4alpha, or GRK6 resulted in an increase in the agonist-induced phosphorylation of the rFSHR. Cotransfections of the rFSHR with GRKs or arrestin-3 enhanced the agonist-induced internalization of the rFHSR, and combinations of GRKs and arrestin-3 were more effective than the individual components. To characterize the involvement of endogenous GRKs on phosphorylation and internalization, we inhibited endogenous GRK2 by overexpression of a kinase-deficient mutant of GRK2 or G alpha t, a scavenger of G betagamma. We also inhibited endogenous GRK6 by overexpression of a kinase-deficient mutant of GKR6. All three constructs were effective inhibitors of phosphorylation, but only the kinase-deficient mutant of GRK2 and G alpha t inhibited internalization. The inhibition of internalization induced by these two constructs was less pronounced than that induced by a dominant-negative mutant of the nonvisual arrrestins, however. The finding that inhibitors of GRK2 and GRK6 impair phosphorylation, but only the inhibitors of GRK2 impair internalization, suggests that different GRKs have differential effects on receptor internalization.     

SDF-1/CXCL12 regulates cAMP production and ion transport in intestinal epithelial cells via CXCR4

Human colonic epithelial cells express CXCR4, the sole cognate receptor for the chemokine stromal cell-derived factor (SDF)-1/CXC chemokine ligand (CXCL) 12. The aim of this study was to define the mechanism and functional consequences of signaling intestinal epithelial cells through the CXCR4 chemokine receptor. CXCR4, but not SDF-1/CXCL12, was constitutively expressed by T84, HT-29, HT-29/-18C1, and Caco-2 human colon epithelial cell lines. Studies using T84 cells showed that CXCR4 was G protein-coupled in intestinal epithelial cells. Moreover, stimulation of T84 cells with SDF-1/CXCL12 inhibited cAMP production in response to the adenylyl cyclase activator forskolin, and this inhibition was abrogated by either anti-CXCR4 antibody or receptor desensitization. Studies with pertussis toxin suggested that SDF-1/CXCL12 activated negative regulation of cAMP production through G(i)alpha subunits coupled to CXCR4. Consistent with the inhibition of forskolin-stimulated cAMP production, SDF-1/CXCL12 also inhibited forskolin-induced ion transport in voltage-clamped polarized T84 cells. Taken together, these data indicate that epithelial CXCR4 can transduce functional signals in human intestinal epithelial cells that modulate important cAMP-mediated cellular functions.