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 Galphai 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 immunofluorescence, endogenous and GFP-tagged GIPC show both a diffuse and punctate cytoplasmic distribution in HeLa cells reflecting, 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.

Figure 1

(A) Alignment of human, rat, mouse and two C. elegans GIPC proteins (C35 and F44). The PDZ domain is underlined, and seven putative protein kinase C (▾) and six casein kinase II (▿) sites are indicated. (B) Sequence alignment of selected PDZ domains. Identical residues are highlighted in deep gray, conserved residues in pale gray. Amino acid numbering corresponds to that of GIPC. The sequences are as follows: CASK (residues 486–518, AF032119), PSD95–PDZ3 (residues 308–401, P78352), DLG–PDZ3 (residues 462–555, Q12959), GRIP–PDZ2 (residues 668–764, U88572), AIP–PDZ3 (residues 601–691, AF038563). The numbers in parenthesis refer to the positions of amino acids and the GenBank accession numbers, respectively.

Figure 2

(A) GIPC interacts specifically with GAIP. (B) GIPC interacts with the C terminus of GAIP. (C) GAIP interacts with the PDZ domain of GIPC. The β-gal filter assay was performed on Leu⁻, Trp⁻ plates, and intensity of color was scored as the following: −−−, no color (background) after 16 hr; +/−, very faint color (but above background after 16 hr); +++, strong color after 2 hr. Yeast cotransformed with void bait and prey vectors were taken as background.

Figure 3

GIPC interacts specifically with GAIP in vitro. GST-fusion proteins bound to glutathione-agarose beads were incubated with in vitro-translated, radiolabeled GIPC as described. GIPC bound specifically to GAIP (lane 3) but not to GST alone (lane 2). GIPC did not bind to RGS4 (lane 4), to the RGS domain of GAIP (GAIP_80–206, lane 5), or the N-terminal domain of GAIP (GAIP_1–79, lane 6). GIPC appears as a 40-kDa doublet (lane 1) in ³⁵S-labeled in vitro-translated GIPC product.

Figure 4

GIPC interacts with the C terminus of GAIP. GST–GAIP fusion protein bound to glutathione-agarose beads was incubated with in vitro-translated GIPC (lane 3) as described. Addition of 30 μg (lane 4) or 300 μg (lane 5) of anti-GAIP C-terminal IgG reduced the binding of GIPC to GAIP, whereas 300 μg anti-CALNUC antibody (lane 6) had minimal effect. When 10 μM (lane 7) or 1 mM (lane 8) of GAIP C-terminal peptide were added the binding of GIPC to GAIP was also reduced 16% and 43%, respectively. A control peptide (1 mM, lane 9) had little effect on binding. Binding to GST alone (lane 2) was taken as background, and the signal obtained after binding of GIPC to GST–GAIP (lane 3) (with background subtracted) was defined as 100% in arbitrary units. Lane 1, ³⁵S-labeled in vitro-translated GIPC product.

Figure 5

Expression and tissue distribution of human GIPC mRNA. The autoradiograph was exposed for 16 hr at −70°C with intensifying screens. hGIPC showed an ubiquitous 1.8-kb transcript with highest expression levels in pancreas, skeletal muscle, kidney, placenta, and brain.

Figure 6

GIPC is present in both membrane and cytosolic fractions. In HeLa cells most of the endogenous GIPC (70%) is found in the soluble (S) or cytosolic fraction, prepared from a postnuclear supernatant (PN), but the remainder (30%) sediments with the membrane pellet. Fractions were immunoblotted with anti-GIPC, antiserum detected by enhanced chemiluminescence, and quantified as described.

Figure 7

Localization of GIPC–GFP and endogenous GIPC in HeLa cells. Phase contrast (A) and fluorescence (B, C, E, and F) micrographs of HeLa cells transiently transfected with GIPC–GFP. Two patterns of staining are seen 20 h after transfection: a punctate (B) and a diffuse (C) cytosolic staining pattern. (D) Endogenous GIPC shows a similar diffuse and punctate vesicular staining pattern in nontransfected HeLa cells by immunofluorescence. (E) When cells expressing GIPC–GFP were stained with affinity purified GAIP IgG, little overlap was seen in the distribution of GIPC-GFP (E) and GAIP (F). In D, nontransfected HeLa cells were aldehyde-fixed and incubated with rabbit polyclonal anti-GIPC antibody followed by Texas Red-conjugated donkey anti-rabbit F(ab′)₂ as described. (×600.)

Figure 8

GIPC is associated with vesicles in HeLa cells. (A and B) Gold particles are found on vesicles (arrows), which are typically arranged in clusters located near the plasma membrane (PM). Some gold particles are also seen scattered throughout the cytoplasm (arrowheads). Cells were processed as described. Ultrathin cryosections were incubated sequentially with rabbit polyclonal anti-GIPC IgG and 5-nm gold, goat anti-rabbit IgG conjugate. (×120,000.)