Browsing by Author "Pitcher, JA"

Now showing 1 - 5 of 5
  • Thumbnail Image
    ItemRestricted
    beta2-Adrenergic receptor regulation by GIT1, a G protein-coupled receptor kinase-associated ADP ribosylation factor GTPase-activating protein.
    (Proc Natl Acad Sci U S A, 1998-11-24) Premont, RT; Claing, A; Vitale, N; Freeman, JL; Pitcher, JA; Patton, WA; Moss, J; Vaughan, M; Lefkowitz, RJ
    G protein-coupled receptor activation leads to the membrane recruitment and activation of G protein-coupled receptor kinases, which phosphorylate receptors and lead to their inactivation. We have identified a novel G protein-coupled receptor kinase-interacting protein, GIT1, that is a GTPase-activating protein (GAP) for the ADP ribosylation factor (ARF) family of small GTP-binding proteins. Overexpression of GIT1 leads to reduced beta2-adrenergic receptor signaling and increased receptor phosphorylation, which result from reduced receptor internalization and resensitization. These cellular effects of GIT1 require its intact ARF GAP activity and do not reflect regulation of GRK kinase activity. These results suggest an essential role for ARF proteins in regulating beta2-adrenergic receptor endocytosis. Moreover, they provide a mechanism for integration of receptor activation and endocytosis through regulation of ARF protein activation by GRK-mediated recruitment of the GIT1 ARF GAP to the plasma membrane.
  • Thumbnail Image
    ItemOpen Access
    Identification of the endophilins (SH3p4/p8/p13) as novel binding partners for the beta1-adrenergic receptor.
    (Proc Natl Acad Sci U S A, 1999-10-26) Tang, Y; Hu, LA; Miller, WE; Ringstad, N; Hall, RA; Pitcher, JA; DeCamilli, P; Lefkowitz, RJ
    Several G-protein coupled receptors, such as the beta1-adrenergic receptor (beta1-AR), contain polyproline motifs within their intracellular domains. Such motifs in other proteins are known to mediate protein-protein interactions such as with Src homology (SH)3 domains. Accordingly, we used the proline-rich third intracellular loop of the beta1-AR either as a glutathione S-transferase fusion protein in biochemical "pull-down" assays or as bait in the yeast two-hybrid system to search for interacting proteins. Both approaches identified SH3p4/p8/p13 (also referred to as endophilin 1/2/3), a SH3 domain-containing protein family, as binding partners for the beta1-AR. In vitro and in human embryonic kidney (HEK) 293 cells, SH3p4 specifically binds to the third intracellular loop of the beta1-AR but not to that of the beta2-AR. Moreover, this interaction is mediated by the C-terminal SH3 domain of SH3p4. Functionally, overexpression of SH3p4 promotes agonist-induced internalization and modestly decreases the Gs coupling efficacy of beta1-ARs in HEK293 cells while having no effect on beta2-ARs. Thus, our studies demonstrate a role of the SH3p4/p8/p13 protein family in beta1-AR signaling and suggest that interaction between proline-rich motifs and SH3-containing proteins may represent a previously underappreciated aspect of G-protein coupled receptor signaling.
  • Thumbnail Image
    ItemOpen Access
    Receptor and G betagamma isoform-specific interactions with G protein-coupled receptor kinases.
    (Proc Natl Acad Sci U S A, 1997-03-18) Daaka, Y; Pitcher, JA; Richardson, M; Stoffel, RH; Robishaw, JD; Lefkowitz, RJ
    The G protein-coupled receptor (GPCR) kinases (GRKs) phosphorylate and desensitize agonist-occupied GPCRs. GRK2-mediated receptor phosphorylation is preceded by the agonist-dependent membrane association of this enzyme. Previous in vitro studies with purified proteins have suggested that this translocation may be mediated by the recruitment of GRK2 to the plasma membrane by its interaction with the free betagamma subunits of heterotrimeric G proteins (G betagamma). Here we demonstrate that this mechanism operates in intact cells and that specificity is imparted by the selective interaction of discrete pools of G betagamma with receptors and GRKs. Treatment of Cos-7 cells transiently overexpressing GRK2 with a beta-receptor agonist promotes a 3-fold increase in plasma membrane-associated GRK2. This translocation of GRK2 is inhibited by the carboxyl terminus of GRK2, a known G betagamma sequestrant. Furthermore, in cells overexpressing both GRK2 and G beta1 gamma2, activation of lysophosphatidic acid receptors leads to the rapid and transient formation of a GRK/G betagamma complex. That G betagamma specificity exists at the level of the GPCR and the GRK is indicated by the observation that a GRK2/G betagamma complex is formed after agonist occupancy of the lysophosphatidic acid and beta-adrenergic but not thrombin receptors. In contrast to GRK2, GRK3 forms a G betagamma complex after stimulation of all three GPCRs. This G betagamma binding specificity of the GRKs is also reflected at the level of the purified proteins. Thus the GRK2 carboxyl terminus binds G beta1 and G beta2 but not G beta3, while the GRK3 fusion protein binds all three G beta isoforms. This study provides a direct demonstration of a role for G betagamma in mediating the agonist-stimulated translocation of GRK2 and GRK3 in an intact cellular system and demonstrates isoform specificity in the interaction of these components.
  • Thumbnail Image
    ItemOpen Access
    The G-protein-coupled receptor phosphatase: a protein phosphatase type 2A with a distinct subcellular distribution and substrate specificity.
    (Proc Natl Acad Sci U S A, 1995-08-29) Pitcher, JA; Payne, ES; Csortos, C; DePaoli-Roach, AA; Lefkowitz, RJ
    Phosphorylation of G-protein-coupled receptors plays an important role in regulating their function. In this study the G-protein-coupled receptor phosphatase (GRP) capable of dephosphorylating G-protein-coupled receptor kinase-phosphorylated receptors is described. The GRP activity of bovine brain is a latent oligomeric form of protein phosphatase type 2A (PP-2A) exclusively associated with the particulate fraction. GRP activity is observed only when assayed in the presence of protamine or when phosphatase-containing fractions are subjected to freeze/thaw treatment under reducing conditions. Consistent with its identification as a member of the PP-2A family, the GRP is potently inhibited by okadaic acid but not by I-2, the specific inhibitor of protein phosphatase type 1. Solubilization of the membrane-associated GRP followed by gel filtration in the absence of detergent yields a 150-kDa peak of latent receptor phosphatase activity. Western blot analysis of this phosphatase reveals a likely subunit composition of AB alpha C. PP-2A of this subunit composition has previously been characterized as a soluble enzyme, yet negligible soluble GRP activity was observed. The subcellular distribution and substrate specificity of the GRP suggests significant differences between it and previously characterized forms of PP-2A.
  • Thumbnail Image
    ItemOpen Access
    Tyrosine phosphorylation of G protein alpha subunits by pp60c-src.
    (Proc Natl Acad Sci U S A, 1992-07-01) Hausdorff, WP; Pitcher, JA; Luttrell, DK; Linder, ME; Kurose, H; Parsons, SJ; Caron, MG; Lefkowitz, RJ
    A number of lines of evidence suggest that cross-talk exists between the cellular signal transduction pathways involving tyrosine phosphorylation catalyzed by members of the pp60c-src kinase family and those mediated by guanine nucleotide regulatory proteins (G proteins). In this study, we explore the possibility that direct interactions between pp60c-src and G proteins may occur with functional consequences. Preparations of pp60c-src isolated by immunoprecipitation phosphorylate on tyrosine residues the purified G-protein alpha subunits (G alpha) of several heterotrimeric G proteins. Phosphorylation is highly dependent on G-protein conformation, and G alpha(GDP) uncomplexed by beta gamma subunits appears to be the preferred substrate. In functional studies, phosphorylation of stimulatory G alpha (G alpha s) modestly increases the rate of binding of guanosine 5'-[gamma-[35S]thio]triphosphate to Gs as well as the receptor-stimulated steady-state rate of GTP hydrolysis by Gs. Heterotrimeric G proteins may represent a previously unappreciated class of potential substrates for pp60c-src.