GIPC, A PDZ DOMAIN-CONTAINING PROTEIN, INTERACTS SPECIFICALLY WITH THE C-TERMINUS OF RGS-GAIP

We have identified a mammalian protein called GIPC (for GAIP interacti ng protein, C terminus), which has a central PDZ domain and a C-termin al acyl carrier protein (ACP) domain. The PDZ domain of GIPC specifica lly interacts with RGS-GAIP, a GTPase-activating protein (GAP) for G a lpha(i) subunits recently localized on clathrin-coated vesicles. Analy sis of deletion mutants indicated that the PDZ domain of GIPC specific ally interacts with the C terminus of GAIP (11 amino acids) in the yea st two-hybrid system and glutathione S-transferase (GST)-GIPC pull-dow n assays, but GIPC does not interact with other members of the RGS (re gulators of G protein signaling) family tested. This finding is in kee ping with the fact that the C terminus of GAIP is unique and possesses a modified C-terminal PDZ-binding motif (SEA). By immunoblotting of m embrane fractions prepared from HeLa cells, we found that there are tw o pools of GIPC-a soluble or cytosolic pool (70%) and a membrane-assoc iated pool (30%). By immunofluorescence, endogenous and GFP-tagged GIP C show both a diffuse and punctate cytoplasmic distribution in HeLa ce lls reflecting, respectively, the existence of soluble and membrane-as sociated pools. By immunoelectron microscopy the membrane pool of GIPC is associated with clusters of vesicles located near the plasma membr ane. These data provide direct evidence that the C terminus of a RGS p rotein is involved in interactions specific for a given RGS protein an d implicates GAIP in regulation of additional functions besides its GA P activity. The location of GIPC together with its binding to GAIP sug gest that GAIP and GIPC may be components of a G protein-coupled signa ling 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.