Src family kinases negatively regulate platelet-derived growth factor alpha receptor-dependent signaling and disease progression

We tested the hypothesis that Src family kinases (SFK) contribute to c-Cbl-mediated degradation of the platelet-derived growth factor (PDGF) alpha receptor (alphaPDGFR). Using either a receptor mutant that does not engage SFKs (F72/74), or cells that that lack SFKs, we found that SFKs contributed to degradation of the alphaPDGFR. Overexpression of c-Cbl also reduced the receptor half-life, but only if the receptor was able to engage SFKs. In cultured cells, prolonging the half-life of the receptor correlated with enhanced signaling and more efficient S phase entry, whereas accelerating receptor degradation had the opposite effect. Consistent with these tissue culture findings, there was a statistically significant increase in the onset of a proliferative retinal disease when animals were injected with cells expressing the F72/74 receptor, as compared with cells expressing the WT receptor. Our findings suggest that SFKs cooperate with c-Cbl to negatively regulate the alphaPDGFR, and that the SFK/c-Cbl suppression of alphaPDGFR output is relevant to the onset and progression of a proliferative disease.     

c-Src but not Fyn promotes proper spindle orientation in early prometaphase

Src family tyrosine kinases (SFKs) participate in mitotic signal transduction events, including mitotic entry, cleavage furrow ingression, and cytokinesis abscission. Although SFKs have been shown to associate with the mitotic spindle, the role of SFKs in mitotic spindle formation remains unclear. Here, we show that c-Src promotes proper spindle orientation in early prometaphase. Src localizes close to spindle poles in a manner independent of Src kinase activity. Three-dimensional analyses showed that Src inhibition induced spindle misorientation, exhibiting a tilting spindle in early prometaphase. Spindle misorientation is frequently seen in SYF cells, which harbor triple knock-out mutations of c-Src, c-Yes, and Fyn, and reintroduction of c-Src but not Fyn into SYF cells rescued spindle misorientation. Spindle misorientation was also observed upon Src inhibition under conditions in which Aurora B was inhibited. Inducible expression of c-Src promoted a properly oriented bipolar spindle, which was suppressed by Src inhibition. Aster formation was severely inhibited in SYF cells upon Aurora B inhibition, which was rescued by reintroduction of c-Src into SYF cells. Furthermore, reintroduction of c-Src facilitated microtubule regrowth from cold-induced depolymerization and accelerated M phase progression. These results suggest that c-Src is involved in spindle orientation through centrosome-mediated aster formation.     

Src family kinase-independent signal transduction and gene induction by leukemia inhibitory factor

Members of the interleukin-6 (IL-6) family of cytokines exert their biological effects via binding to their cognate ligand-binding receptor subunit on a target cell. The subsequent recruitment of the common signal transducer glycoprotein 130 and activation of the JAK/STAT and SHP-2/Ras/mitogen-activated protein kinase (MAPK) pathways are responsible for the majority of cellular responses elicited by IL-6 cytokines. Several types of experiments suggest that the Src family of kinases (SFK) also participates in IL-6 family cytokine-mediated signaling events. SYF cells, which lack expression of SFKs Src, Yes, and Fyn, were used to determine the role of SFKs in IL-6 family cytokine signaling and gene induction. SYF and wild type (WT) control fibroblasts displayed similar activation of signaling intermediates following stimulation with leukemia inhibitory factor (LIF). LIF-stimulated tyrosine phosphorylation of SHP-2 and subsequent activation of MAPK in SYF cells were identical to that seen in LIF-stimulated WT cells. Both LIF-stimulated tyrosine phosphorylation of STAT1 and STAT3, as well as LIF-stimulated DNA binding activity of STAT-containing nuclear complexes were indistinguishable when compared in SYF and WT cells. In addition, the phosphatidylinositol 3-kinase-sensitive Akt kinase and p38 MAPK were activated by LIF in both SYF and WT cells. Furthermore, LIF-stimulated expression of c-fos, egr-1, and suppressor of cytokine signaling-3 was retained in SYF cells. The IL-6 family cytokine oncostatin M was also capable of activating MAPK, STAT3, STAT1, Akt, and p38 in both WT and SYF cells. These results demonstrate that IL-6 family cytokines can activate a full repertoire of signaling pathways and induce gene expression independent of SFKs.     

c-Src regulates clathrin adapter protein 2 interaction with beta-arrestin and the angiotensin II type 1 receptor during clathrin- mediated internalization

Beta-arrestins are multifunctional adapters involved in the internalization and signaling of G protein-coupled receptors (GPCRs). They target receptors to clathrin-coated pits (CCPs) through binding with clathrin and clathrin adapter 2 (AP-2) complex. They also act as transducers of signaling by recruiting c-Src kinase to certain GPCRs. Here we sought to determine whether c-Src regulates the recruitment of AP-2 to beta-arrestin and the angiotensin II (Ang II) type 1 receptor (AT1R) during internalization. We show that the agonist stimulation of native AT1R in vascular smooth muscle cells (VSMCs) induces the formation of an endogenous complex containing c-Src, beta-arrestins and AP-2. In vitro studies using coimmunoprecipitation experiments and a yeast three-hybrid assay reveal that c-Src stabilizes the agonist-independent association between beta-arrestin2 and the beta-subunit of AP-2 independently of the kinase activity of c-Src. However, although c-Src expression promoted the rapid dissociation of AP-2 from both beta-arrestin and AT1R after receptor stimulation, a kinase-inactive mutant of c-Src failed to induce the dissociation of AP-2 from the agonist-occupied receptor. Thus, the consequence of c-Src in regulating the dissociation of AP-2 from the receptor was also examined on the internalization of AT1R by depleting c-Src in human embryonic kidney (HEK) 293 cells using a small interfering RNA strategy. Experiments in c-Src depleted cells reveal that AT1R remained mostly colocalized with AP-2 at the plasma membrane after Ang II stimulation, consistent with the observed delay in receptor internalization. Moreover, coimmunoprecipitation experiments in c-Src depleted HEK 293 cells and VSMCs showed an increased association of AP-2 to the agonist-occupied AT1R and beta-arrestin, respectively. Together, our results support a role for c-Src in regulating the dissociation of AP-2 from agonist-occupied AT1R and beta-arrestin during the clathrin-mediated internalization of receptors and suggest a novel function for c-Src kinase in the internalization of AT1R.     

Src family kinases are required for integrin but not PDGFR signal transduction

Src family kinases (SFKs) have been implicated as important regulators of ligand-induced cellular responses including proliferation, survival, adhesion and migration. Analysis of SFK function has been impeded by extensive redundancy between family members. We have generated mouse embryos harboring functional null mutations of the ubiquitously expressed SFKs Src, Yes and Fyn. This triple mutation leads to severe developmental defects and lethality by E9.5. To elucidate the molecular mechanisms underlying this phenotype, SYF cells (deficient for Src, Yes and Fyn) were derived and tested for their ability to respond to growth factors or plating on extracellular matrix. Our studies reveal that while Src, Yes and Fyn are largely dispensable for platelet-derived growth factor (PDGF)-induced signaling, they are absolutely required to mediate specific functions regulated by extracellular matrix proteins. Fibronectin-induced tyrosine phosphorylation of focal adhesion proteins, including the focal adhesion kinase FAK, was nearly eliminated in the absence of Src, Yes and Fyn. Furthermore, consistent with previous reports demonstrating the importance of FAK for cell migration, SYF cells displayed reduced motility in vitro. These results demonstrate that SFK activity is essential during embryogenesis and suggest that defects observed in SYF triple mutant embryos may be linked to deficiencies in signaling by extracellular matrix-coupled receptors.     

Regulation of the neuronal nicotinic acetylcholine receptor by SRC family tyrosine kinases

Src family kinases (SFKs) are abundant in chromaffin cells that reside in the adrenal medulla and respond to cholinergic stimulation by secreting catecholamines. Our previous work indicated that SFKs regulate acetylcholine- or nicotine-induced secretion, but the site of modulatory action was unclear. Using whole cell recordings, we found that inhibition of SFK tyrosine kinase activity by PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo(3,4-d)pyrimidine) treatment or expression of a kinase-defective c-Src reduced the peak amplitude of nicotine-induced currents in chromaffin cells or in human embryonic kidney cells ectopically expressing functional neuronal alpha3beta4alpha5 acetylcholine receptors (AChRs). Conversely, the phosphotyrosine phosphatase inhibitor, sodium vanadate, or expression of mutationally activated c-Src resulted in enhanced current amplitudes. These results suggest that SFKs and putative phosphotyrosine phosphatases regulate the activity of AChRs by opposing actions. This proposed model was supported further by the findings that SFKs physically associate with the receptor and that the AChR is tyrosine-phosphorylated.     

Evidence that constitutively active luteinizing hormone/human chorionic gonadotropin receptors are rapidly internalized.

The luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptor, which belongs to the family of G-protein coupled receptors, plays an important role in gonadal steroidogenesis. Substitution of aspartic acid 556 of the LH/hCG receptor with glycine (D556G) creates a constitutively active receptor that activates adenylyl cyclase in the absence of hormone. To examine receptor internalization, human embryonic kidney cells (293 T) expressing wild type (WT) or D556G mutant receptors were incubated with [125I]hCG and subsequently analyzed for cell surface bound and internalized radioactivity. Comparison of the rate constants of internalization of the D556G mutant and WT receptors revealed that the rate of internalization of the D556G mutant was five times greater than that of the WT receptor. Although the D556G receptor internalizes [125I]hCG rapidly, a corresponding increase in [125I]hCG degradation was not seen. The internalization of another constitutively active LH/hCG receptor (aspartic acid 556 to tyrosine) was also greater than that of the WT receptor. Internalization of receptor bound [125I]hCG was inhibited by a hypertonic sucrose solution, confirming that the ligand enters the cell by receptor-mediated endocytosis. Furthermore, the constitutively active D556G and D556Y LH/hCG receptors utilize the arrestin dependent internalization pathway. These results suggest that the active state conformation of the constitutively active receptor is conducive to rapid internalization.     

Muscarinic M2 receptors mediate transactivation of EGF receptor through Fyn kinase and without matrix metalloproteases

Transactivation of epidermal growth factor receptor (EGFR) by G protein-coupled receptors (GPCRs) has been attributed to the activation of matrix metalloproteases (MMPs) and the release of EGF family ligands such as HB-EGF. This mode of transactivation leads to signalling downstream of EGFR which is indistinguishable from that induced by the ligand. Here we provide evidence that in the COS-7 cell model EGFR transactivation via the muscarinic M2 receptor (M2R) is independent of MMPs and results in an incomplete EGFR signalling including ERK and Akt but not PLCgamma1. Using dominant-negative mutants of c-Src and Fyn and Src-deficient SYF cells as well as by co-immunoprecipitation studies, we can demonstrate that the M2R-mediated transactivation of EGFR specifically involves Fyn but not c-Src or Yes. This specific role of Fyn can be verified in SH-SY5Y human neuroblastoma cells with endogenously expressed M2 receptors.     

Rapid agonist-induced desensitization and internalization of the A(2B) adenosine receptor is mediated by a serine residue close to the COOH terminus

The G(s)-coupled rat A(2B) adenosine receptor (A(2B)-AR) was epitope-tagged at the NH(2) terminus with hemagglutinin (HA) and subjected to progressive deletions or point mutations of the COOH terminus in order to determine regions of the receptor that contribute to agonist-induced desensitization and internalization. When expressed stably in Chinese hamster ovary cells, a mutant receptor in which the final 2 amino acids were deleted, the Leu(330)-stop mutant, underwent rapid agonist-induced desensitization and internalization as did the wild type (WT) receptor. However, the Phe(328) and the Gln(325)-stop mutants were resistant to rapid agonist-induced desensitization and internalization. Co-expression of arrestin-2-green fluorescent protein (arrestin-2-GFP) with WT receptor or Leu(330)-stop mutant resulted in rapid translocation of arrestin-2-GFP from cytosol to membrane upon agonist addition. On the other hand, agonist activation of the Phe(328)-stop or Gln(325)-stop mutant did not result in translocation of arrestin-2-GFP from cytosol. A COOH terminus point mutant, S329G, was also unable to undergo rapid agonist-induced desensitization and internalization, indicating that Ser(329) is a critical residue for these processes. A further deletion mutant (Ser(326)-stop) unexpectedly underwent rapid agonist-induced desensitization and internalization. However, activation of this mutant did not promote translocation of arrestin-2-GFP from cytosol to membrane. In addition, whereas WT receptor internalization was markedly inhibited by co-expression of dominant negative mutants of arrestin-2 (arrestin-2-(319-418)), dynamin (dynamin K44A), or Eps-15 (EDelta95-295), Ser(326)-stop receptor internalization was only inhibited by dominant negative mutant dynamin. Taken together these results indicate that Ser(329), close to the COOH terminus of the rat A(2B)-AR, is critical for the rapid agonist-induced desensitization and internalization of the receptor. However, deletion of the COOH terminus also uncovers a motif that is able to redirect internalization of the receptor to an arrestin- and clathrin-independent pathway.     

The platelet-derived growth factor receptor-beta phosphorylates and activates G protein-coupled receptor kinase-2. A mechanism for feedback inhibition

G protein-coupled receptor kinase-2 (GRK2) serine-phosphorylates the platelet-derived growth factor receptor-beta (PDGFRbeta), and thereby diminishes signaling by the receptor. Because activation of GRK2 may involve phosphorylation of its N-terminal tyrosines by c-Src, we tested whether the PDGFRbeta itself could tyrosine-phosphorylate and activate GRK2. To do so, we used wild type (WT) and Y857F mutant PDGFRbetas in HEK cells, which lack endogenous PDGFRs. The Y857F PDGFRbeta autophosphorylates normally but does not phosphorylate exogenous substrates. Although PDGF-stimulated Y857F and WT PDGFRbetas activated c-Src equivalently, the WT PDGFRbeta tyrosine-phosphorylated GKR2 60-fold more than the Y857F PDGFRbeta in intact cells. With purified GRK2 and either WT or Y857F PDGFRbetas immunoprecipitated from HEK cells, GRK2 tyrosyl phosphorylation was PDGF-dependent and required the WT PDGFRbeta, even though the WT and Y857F PDGFRbetas autophosphorylated equivalently. This PDGFRbeta-mediated GRK2 tyrosyl phosphorylation enhanced GRK2 activity: GRK2-mediated seryl phosphorylation of the PDGFRbeta was 9-fold greater for the WT than for the Y857F in response to PDGF, but equivalent when GRK2 was activated by sequential stimulation of beta2-adrenergic and PDGF-beta receptors. Furthermore, both PDGFRbeta-mediated GRK2 tyrosyl phosphorylation and GRK2-mediated PDGFRbeta seryl phosphorylation were reduced approximately 50% in intact cells by mutation to phenylalanine of three tyrosines in the N-terminal domain of GRK2. We conclude that the activated PDGFRbeta itself phosphorylates GRK2 tyrosyl residues and thereby activates GRK2, which then serine-phosphorylates and desensitizes the PDGFRbeta.     

Palmitoylation of the luteinizing hormone/human chorionic gonadotropin receptor regulates receptor interaction with the arrestin-mediated internalization pathway.

The luteinizing hormone/human chorionic gonadotropin receptor (LH/hCGR) undergoes palmitoylation at cysteine residues 621 and 622 located in the carboxyl terminal tail of the receptor. This study examined the biological function of palmitoylation with respect to its effect on receptor internalization. Coexpression of wild-type (WT) or C621/622G mutant receptors with arrestin-2 increased receptor internalization in 293T cells. Furthermore, measurements of rate enhancement upon overexpression of arrestin indicate that the palmitoylation deficient mutant receptor is more prone to utilizing the arrestin mediated internalization pathway than the WT receptor. Coexpression of G-protein-coupled receptor kinase 4 (GRK4) with wild type receptor resulted in an increase in internalization, while coexpression with the mutant receptor did not result in further enhancement of internalization. Additionally, 293T cells expressing mutant receptor were responsive to hCG with respect to production of inositol phosphates. Taken together, these results suggest that the palmitoylation state of the receptor governs internalization by regulating the accessibility of the receptor to the arrestin-mediated internalization pathway.     

Identification of the receptor-associated signaling enzymes that are required for platelet-derived growth factor-AA-dependent chemotaxis and DNA synthesis.

Activation of the platelet-derived growth factor (PDGF) alpha receptor (alphaPDGFR) leads to cell migration and DNA synthesis. These events are preceded by the ligand-induced tyrosine phosphorylation of the receptor and its association with SH2-containing signaling enzymes including Src family members (Src), the phosphotyrosine phosphatase SHP-2, phosphatidylinositol 3-kinase (PI3K), and phospholipase C-gamma1 (PLCgamma). In this study, we sought to systematically evaluate the relative roles of the signaling enzymes that are recruited to the alphaPDGFR for DNA synthesis and cell migration. Our approach was to generate and characterize tyrosine to phenylalanine alphaPDGFR mutants that failed to associate with one or more of the above listed signaling enzymes. In a 3T3-like cell line (Ph cells), PDGF-dependent DNA synthesis was strictly dependent on only one of the receptor-associated proteins, PI3K. In contrast, multiple signaling enzymes were required for maximal chemotaxis, as receptors unable to associate with either Src, PI3K, or PLCgamma initiated chemotaxis to 4, 47, or 56% of the wild-type level, respectively. Furthermore, coexpression of mutant receptors revealed that these signaling enzymes do not need to be on the same receptor for a cell to respond chemotactically to PDGF. We conclude that for the alphaPDGFR, PI3K plays a major role in initiating DNA synthesis, whereas PI3K, PLCgamma, and especially Src are required for chemotaxis.     

Differential roles of the NPXXY motif in formyl peptide receptor signaling

The NPXXY motif (X represents any amino acid) in the seventh transmembrane domain of the chemotactic formyl peptide receptor (FPR) is highly conserved among G protein-coupled receptors. Recent work suggested that this motif contributes to G protein-coupled receptor internalization and signal transduction; however, its role in FPR signaling remains unclear. In this study we replaced Asn(297) and Tyr(301) in the NPXXY motif of the human FPR with Ala (N297A) and Ala/Phe (Y301A/Y301F), respectively, and determined the effects of the substitutions on FPR functions in transfected rat basophilic leukemia cells. Whereas all the mutant receptors were expressed on the cell surface, the N297A receptor exhibited reduced binding affinity and was unable to mediate activation of phospholipase C-beta and the p42/44 mitogen-activated protein kinase (MAP kinase). The Y301F receptor displayed significantly decreased ligand-stimulated internalization and MAP kinase activation, suggesting that the hydrogen bonding at Tyr(301) is critical for these functions. The Y301F receptor showed a chemotactic response similar to that of wild-type FPR, indicating that cell chemotaxis does not require receptor internalization and hydrogen bonding at the Tyr(301) position. In contrast, the Y301A receptor displayed a left-shifted, but overall reduced, chemotaxis response that peaked at 0.1-1 nM. Finally, using a specific MAP kinase kinase inhibitor, we found that activation of MAP kinase is required for efficient FPR internalization, but is not essential for chemotaxis. These findings demonstrate that residues within the NPXXY motif differentially regulate the functions of FPR.     

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.     

Arrestins block G protein-coupled receptor-mediated apoptosis

G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptors (GPCRs) activate numerous cellular signals through the combined actions of G proteins, GPCR kinases, and arrestins. Although arrestins have traditionally been thought of as mediating GPCR desensitization, they have now been shown to play important roles in the internalization, trafficking, and signaling of many GPCRs. We demonstrate that in cells devoid of arrestins, the stimulation of numerous GPCRs including the N-formyl peptide receptor (FPR) initiates rapid cell rounding, annexin V positivity, and caspase activation followed by cell death. The apoptotic response is initiated by G protein signaling and involves activation of phosphoinositide 3-kinase, mitogen-activated protein kinases, and c-Src resulting in cytochrome c release from mitochondria and ultimately caspase 9 and caspase 3 activation. Reconstitution with either arrestin-2 or arrestin-3 is completely sufficient to prevent FPR-mediated apoptosis. Surprisingly, a non-desensitizing and non-internalizing mutant of the FPR is unable to initiate apoptosis, indicating that receptor phosphorylation and internalization, but not solely chronic activation due to a lack of desensitization, are critical determinants for the induction of apoptosis by the FPR. We further demonstrate that this response is not unique to the FPR with numerous additional GPCRs, including the V2 vasopressin, angiotensin II (type 1A), and CXCR2 receptors, capable of initiating apoptosis upon stimulation, whereas GPCRs such as the beta(2)-adrenergic receptor and CXCR4 are not capable of initiating apoptotic signaling. These data demonstrate for the first time that arrestins play a critical and completely unexpected role in the suppression GPCR-mediated apoptosis, which we show is a common consequence of GPCR-mediated cellular activation in the absence of arrestins.     

Distinct conformations of the corticotropin releasing factor type 1 receptor adopted following agonist and antagonist binding are differentially regulated

The corticotropin releasing factor (CRF) type 1 receptor (CRF1) is a class B family G protein-coupled receptor that regulates the hypothalamic-pituitary-adrenal stress axis. Astressin is an amino-terminal truncated analog of CRF that retains high affinity binding to the extracellular domain of the receptor and is believed to act as a neutral competitive antagonist of receptor activation. Here we show that despite being unable to activate the CRF1 receptor, astressin binding results in the internalization of the receptor. Furthermore, entirely different pathways of internalization of CRF1 receptors are utilized following CRF and astressin binding. CRF causes the receptor to be phosphorylated, recruit beta-arrestin2, and to be internalized rapidly, likely through clathrin-coated pits. Astressin, however, fails to induce receptor phosphorylation or beta-arrestin2 recruitment, and internalization is slow and occurs through a pathway that is insensitive to inhibitors of clathrin-coated pits and caveolae. The fate of the internalized receptors also differs because only CRF-induced internalization results in receptor down-regulation. Furthermore, we present evidence that for astressin to induce internalization it must interact with both the extracellular amino terminus and the juxtamembrane domain of the receptor. Astressin binds with 6-fold higher affinity to full-length CRF1 receptors than to a chimeric protein containing only the extracellular domain attached to the transmembrane domain of the activin IIB receptor, yet two 12-residue analogs of astressin have similar affinities for both proteins but are unable to induce receptor internalization. These data demonstrate that agonists and antagonists for CRF1 receptors promote distinct conformations, which are then differentially regulated.     

Phosphorylation of tyrosine 720 in the platelet-derived growth factor alpha receptor is required for binding of Grb2 and SHP-2 but not for activation of Ras or cell proliferation.

Following binding of platelet-derived growth factor (PDGF), the PDGF alpha receptor (alphaPDGFR) becomes tyrosine phosphorylated and associates with a number of signal transduction molecules, including phospholipase Cgamma-1 (PLCgamma-1), phosphatidylinositol 3-kinase (PI3K), the phosphotyrosine phosphatase SHP-2, Grb2, and Src. Here, we present data identifying a novel phosphorylation site in the kinase insert domain of the alphaPDGFR at tyrosine (Y) 720. We replaced this residue with phenylalanine and expressed the mutated receptor (F720) in Patch fibroblasts that do not express the alphaPDGFR. Characterization of the F720 mutant indicated that binding of two proteins, SHP-2 and Grb2, was severely impaired, whereas PLCgamma-1 and PI3K associated to wild-type levels. In addition, mutating Y720 to phenylalanine dramatically reduced PDGF-dependent tyrosine phosphorylation of SHP-2. Since Y720 was required for recruitment of two proteins, we investigated the mechanism by which these two proteins associated with the alphaPDGFR. SHP-2 bound the alphaPDGFR directly, whereas Grb2 associated indirectly, most probably via SHP-2, as Grb2 and SHP-2 coimmunoprecipitated when SHP-2 was tyrosine phosphorylated. We also compared the ability of the wild-type and F720 alphaPDGFRs to mediate a number of downstream events. Preventing the alphaPDGFR from recruiting SHP-2 and Grb2 did not compromise PDGF-AA-induced activation of Ras, initiation of DNA synthesis, or growth of cells in soft agar. We conclude that phosphorylation of the alphaPDGFR at Y720 is required for association of SHP-2 and Grb2 and tyrosine phosphorylation of SHP-2; however, these events are not required for the alphaPDGFR to activate Ras or initiate a proliferative response. In addition, these findings reveal that while SHP-2 binds to both of the receptors, it binds in different locations: to the carboxy terminus of the betaPDGFR but to the kinase insert of the alphaPDGFR.     

ATP binding site mutagenesis reveals different subunit stoichiometry of functional P2X2/3 and P2X2/6 receptors

The aim of the present experiments was to clarify the subunit stoichiometry of P2X2/3 and P2X2/6 receptors, where the same subunit (P2X2) forms a receptor with two different partners (P2X3 or P2X6). For this purpose, four non-functional Ala mutants of the P2X2, P2X3, and P2X6 subunits were generated by replacing single, homologous amino acids particularly important for agonist binding. Co-expression of these mutants in HEK293 cells to yield the P2X2 WT/P2X3 mutant or P2X2 mutant/P2X3 WT receptors resulted in a selective blockade of agonist responses in the former combination only. In contrast, of the P2X2 WT/P2X6 mutant and P2X2 mutant/P2X6 WT receptors, only the latter combination failed to respond to agonists. The effects of α,β-methylene-ATP and 2-methylthio-ATP were determined by measuring transmembrane currents by the patch clamp technique and intracellular Ca(2+) transients by the Ca(2+)-imaging method. Protein labeling, purification, and PAGE confirmed the assembly and surface trafficking of the investigated WT and WT/mutant combinations in Xenopus laevis oocytes. In conclusion, both electrophysiological and biochemical investigations uniformly indicate that one subunit of P2X2 and two subunits of P2X3 form P2X2/3 heteromeric receptors, whereas two subunits of P2X2 and one subunit of P2X6 constitute P2X2/6 receptors. Further, it was shown that already two binding sites of the three possible ones are sufficient to allow these receptors to react with their agonists.     

Complement component 5a receptor oligomerization and homologous receptor down-regulation

Most G-protein-coupled receptors (GPCRs) form di(oligo)-meric structures that constitute signaling and trafficking units and might be essential for receptor functions. Cell responses to complement C5a receptor (C5aR) are tightly controlled by receptor desensitization and internalization. To examine the implication of dimerization in C5aR regulation, we generated an NH(2)-terminally modified C5aR mutant, unable to bind C5a, and a phosphorylation-deficient mutant. Neither an intact NH(2) terminus nor the presence of COOH-terminal phosphorylation sites appeared to be required for the formation of C5aR dimers. Upon C5a stimulation, mutant receptors did not internalize when individually expressed. C5a stimulation of cells that co-expressed wild type C5aR together with either unliganded or phosphorylation-deficient mutant resulted in co-internalization of mutant receptors with C5aR. Unliganded GPCRs can be cross-phosphorylated within a heterologous receptor dimer or by second messenger-activated kinases. C5a stimulation of (32)P-labeled cells that co-expressed the unliganded mutant with either C5aR or the phosphorylation-deficient mutant did not induce phosphorylation of the unliganded mutant. We can thus postulate that, in the case of C5aR, the stimulation and phosphorylation of one monomer is enough to lead to dimer internalization. The existence and functional implication of di(oligo)mer formation may be important for an accurate C5aR down-regulation in pathological conditions.     

EGF transregulates opioid receptors through EGFR-mediated GRK2 phosphorylation and activation

G protein-coupled receptor (GPCR) kinases (GRKs) are key regulators of GPCR function. Here we demonstrate that activation of epidermal growth factor receptor (EGFR), a member of receptor tyrosine kinase family, stimulates GRK2 activity and transregulates the function of G protein-coupled opioid receptors. Our data showed that EGF treatment promoted DOR internalization induced by DOR agonist and this required the intactness of GRK2-phosphorylation sites in DOR. EGF stimulation induced the association of GRK2 with the activated EGFR and the translocation of GRK2 to the plasma membrane. After EGF treatment, GRK2 was phosphorylated at tyrosyl residues. Mutational analysis indicated that EGFR-mediated phosphorylation occurred at GRK2 N-terminal tyrosyl residues previously shown as c-Src phosphorylation sites. However, c-Src activity was not required for EGFR-mediated phosphorylation of GRK2. In vitro assays indicated that GRK2 was a direct interactor and a substrate of EGFR. EGF treatment remarkably elevated DOR phosphorylation in cells expressing the wild-type GRK2 in an EGFR tyrosine kinase activity-dependent manner, whereas EGF-stimulated DOR phosphorylation was greatly decreased in cells expressing mutant GRK2 lacking EGFR tyrosine kinase sites. We further showed that EGF also stimulated internalization of mu-opioid receptor, and this effect was inhibited by GRK2 siRNA. These data indicate that EGF transregulates opioid receptors through EGFR-mediated tyrosyl phosphorylation and activation of GRK2 and propose GRK2 as a mediator of cross-talk from RTK to GPCR signaling pathway.