GIPC, a PDZ domain containing protein, interacts specifically with the C terminus of RGS-GAIP

Abstract

We have identified a mammalian protein called GIPC (for GAIP interacting protein, C terminus), which has a central PDZ domain and a C-terminal acyl carrier protein (ACP) domain. The PDZ domain of GIPC specifically interacts with RGS-GAIP, a GTPase-activating protein (GAP) for G␣ i subunits recently localized on clathrin-coated vesicles. Analysis of deletion mutants indicated that the PDZ domain of GIPC specifically interacts with the C terminus of GAIP (11 amino acids) in the yeast two-hybrid system and glutathione S-transferase (GST)-GIPC pull-down assays, but GIPC does not interact with other members of the RGS (regulators of G protein signaling) family tested. This finding is in keeping with the fact that the C terminus of GAIP is unique and possesses a modified C-terminal PDZ-binding motif (SEA). By immunoblotting of membrane fractions prepared from HeLa cells, we found that there are two pools of GIPC-a soluble or cytosolic pool (70%) and a membrane-associated pool (30%). By immunof luorescence, endogenous and GFP-tagged GIPC show both a diffuse and punctate cytoplasmic distribution in HeLa cells ref lecting, respectively, the existence of soluble and membrane-associated pools. By immunoelectron microscopy the membrane pool of GIPC is associated with clusters of vesicles located near the plasma membrane. These data provide direct evidence that the C terminus of a RGS protein is involved in interactions specific for a given RGS protein and implicates GAIP in regulation of additional functions besides its GAP activity. The location of GIPC together with its binding to GAIP suggest that GAIP and GIPC may be components of a G protein-coupled signaling complex involved in the regulation of vesicular trafficking. The presence of an ACP domain suggests a putative function for GIPC in the acylation of vesicle-bound proteins.The recently discovered family of RGS proteins (for regulators of G protein signaling) act as GTPase-activating proteins (GAPs) that bind to ␣ subunits of heterotrimeric G proteins of the G␣ i ͞G␣ q subclass and enhance their GTPase activity (1-5), thus facilitating their deactivation. Moreover, some family members serve as effector antagonists that compete with effector for binding to G␣ subunits (4).Today more than 20 different mammalian proteins are known to contain the diagnostic RGS domain, far more than there are G␣ i ͞G␣ q subunits. Although their RGS domains are highly homologous (45-80%), the N and C termini of mammalian RGS proteins outside the common RGS domain share little sequence similarity, suggesting that they have specific interactions and functions.Previously we have identified and characterized RGS-GAIP (6, 7), a RGS family member shown to directly interact with G␣ i3 through its RGS domain (6). We subsequently showed that GAIP is membrane-anchored (7) and, quite recently, that it is located on clathrin-coated vesicles and not on the plasma membrane (8). The localization of GAIP to clathrin-coated vesicles suggests GAIP may participate in the regulation of G protein-me...

The RGSZ2 Protein Exists in a Complex with μ-Opioid Receptors and Regulates the Desensitizing Capacity of Gz Proteins

“…Besides the RGS box and a short C terminus of 11 or 12 amino-acid residues, these proteins contain a heavily palmitoylated cysteine string motif at the N terminus that is involved in membrane targeting (De Vries et al, 1996). GAIP also has an amphipathic helix at the N terminus and a PDZbinding motif at the C terminus (De Vries et al, 1998;Ross and Wilkie, 2000). The RGSZ2 described by Jordan et al (1999) lacks the amphipathic helix at the N terminus and the C-terminal PDZ-binding motif present in GAIP, exhibiting closer homology to RGSZ1.…”

RGSZ1 and GAIP Regulate μ- but Not δ-Opioid Receptors in Mouse CNS: Role in Tachyphylaxis and Acute Tolerance

“…GAIP also has an amphipathic helix at the N terminus and a PDZ binding motif at the C terminus (De Vries et al, 1998a;Ross and Wilkie, 2000). The PDZ-binding motif of the GAIP C terminus interacts specifically with the PDZ domain of GIPC (GAIP interacting protein C-terminus) (De Vries et al, 1998b). This CNS protein does not interact with other RGS proteins and, like other PDZ domain proteins, serves to cluster transmembrane receptors (and probably other proteins) with signaling molecules (Lou et al, 2001).…”

GIPC Recruits GAIP (RGS19) To Attenuate Dopamine D2Receptor Signaling

“…A previous study (De Vries et al, 1998b) also showed that full-length RGS2, RGS4, RGS16, and RET-RGS did not interact with GIPC. Interaction between GIPC and GAIP was based on a C-terminal PDZ-recognition motif (De Vries et al, 1998b), in which the C-terminal alanine residue is critical because its deletion broke the interaction (Table 1). Moreover, GIPC binds to several GPCRs, including the dopamine D 2 R, D 3 R, ␤1-adrenergic (␤ 1 AR) and LH (LHR) receptors, through different types of PDZ-binding motifs (Table 1, underlined motifs).…”

“…The PDZ-domain-containing protein GIPC was identified by virtue of its interaction with GAIP, a member of the RZ RGS subfamily (De Vries et al, 1998b). GIPC was recently demonstrated to interact with GPCRs, such as the dopamine D 2 R and D 3 R (Jeanneteau et al, 2004) and ␤1-adrenergic receptors (Hu et al, 2003), raising the possibility that GIPC may serve as a molecular adaptor between GPCR and RGS.…”

“…The aim of this study was to examine the contribution of GIPC, a scaffold protein, in the assembly of a specific RGS-GPCR complex in living cells and the functional role of this assembly on GPCR-mediated G protein signaling. Specifically, we have studied the complex formed with GAIP (RGS19) and D 2 R, which both selectively interact with GIPC (De Vries et al, 1998b;Jeanneteau et al, 2004). The PDZ-domain of GIPC that mediates interactions with GAIP and GPCR has the particularity to bind to either class of PDZ-binding motifs, but to discriminate closely related PDZ-binding consensus.…”