Clathrin-coated vesicles bearing GAIP possess GTPase-activating protein activity in vitro

Abstract

G␣-interacting protein (GAIP) is a member of the RGS (regulators of G protein signaling) family, which serve as GAPs (GTPase-activating proteins) for G␣ subunits. Previously, we demonstrated that GAIP is localized on clathrin-coated vesicles (CCVs). Here, we tested whether GAIPenriched vesicles could accelerate the GTPase activity of G␣ i proteins. A rat liver fraction containing vesicular carriers (CV2) was enriched (4.5؋) for GAIP by quantitative immunoblotting, and GAIP was detected on some of the vesicles in the CV2 fraction by immunoelectron microscopy. When liver fractions were added to recombinant G␣ i3 and tested for GAP activity, only the CV2 fraction contained GAP activity. Increasing amounts of CV2 increased the activity, whereas immunodepletion of the CV2 fraction with an antibody against the C terminus of GAIP decreased GAP activity. CCV fractions were prepared from rat liver by using a protocol that maintains the clathrin coats. GAIP was enriched in these fractions and was detected on CCVs by immunogold labeling. Addition of increasing amounts of CCV to recombinant G␣ i3 protein increased the GTPase activity. We conclude that CCVs possess GAP activity for G␣ i3 and that membrane-associated GAIP is capable of interacting with G␣ i3 . The reconstitution of the interaction between a heterotrimeric G protein and GAIP on CCVs provides biochemical evidence for a model whereby the G protein and its GAP are compartmentalized on different membranes and come into contact at the time of vesicle fusion. Alternatively, they may be located on the same membrane and segregate at the time of vesicle budding.

GIPC Recruits GAIP (RGS19) To Attenuate Dopamine D2Receptor Signaling

“…Indeed, RGS9 -2 accelerates the off-kinetics of D 2 R-induced GIRK currents (Rahman et al, 2003), whereas GAIP was here found to participate in the regulation of adenylate cyclase and phospholipase A2 signaling cascades as well as in vesicular trafficking in accordance with previous studies (Lou et al, 2002;Wylie et al, 2003). In agreement with this latter observation, GAIP and GIPC were closely associated with clathrin as assessed by electron microscopy (De Vries et al, 1998a;Fischer et al, 1999). Additionally, we showed that D 2 R, GIPC and GAIP colocalized within microdomains of the PM, probably clathrin-coated pits, where GFPGAIP codistributed upon D 2 R activation.…”

Promotion of Gαi3 subunit down-regulation by GIPN, a putative E3 ubiquitin ligase that interacts with RGS-GAIP

“…GIPN was cloned into pcDNA3 (Invitrogen) with an N-terminal FLAG-tag and prepared by using the TnT reticulocyte lysate kit (Promega) in the presence of [ 35 S]methionine. Five micrograms of His6-GAIP (17) and AGS3 (residues 424-650; ref. 18) were immobilized on Ni 2ϩ -agarose beads (Qiagen, Valencia, CA) and incubated with 35 S-labeled GIPN for 1 h at 4°C in 300 l of buffer A (20 mM Hepes, pH 7.4͞150 mM NaCl͞0.5 mM EDTA͞0.1% Triton X-100͞0.1 g/ l BSA) with gentle rocking.…”

Membrane-associated GAIP is a phosphoprotein and can be phosphorylated by clathrin-coated vesicles

“…13 and 14). We localized one member of the RGS family, RGS-GAIP (G ␣ interacting protein), by immunocytochemistry and found it to be associated with clathrincoated pits and vesicles (15,16) and further demonstrated that CCVs isolated from rat liver possess GAP activity (16).…”

“…13 and 14). We localized one member of the RGS family, RGS-GAIP (G ␣ interacting protein), by immunocytochemistry and found it to be associated with clathrincoated pits and vesicles (15,16) and further demonstrated that CCVs isolated from rat liver possess GAP activity (16).Clathrin-coated vesicles (CCVs) are known to play a role in G-protein coupled receptor-(2) and receptor-mediated endocytosis at the plasma membrane and in sorting of lysosomal enzymes in the trans-Golgi network (TGN) (17)(18)(19). Formation of CCVs is known to be tightly regulated by phosphorylation and dephosphorylation of adapters and coat proteins (20)(21)(22)(23)(24).…”

“…Residual microsomes (RM) and CCV-enriched fractions were obtained from rat liver as described (15,16). A total of 3.5 or 7 l RM fraction (14 mg͞ml) was resuspended in 100 l of CIP buffer [50 mM Tris⅐HCl (pH 9)͞1 mM EDTA͞100 mM ZnCl 2 ͞1 mM spermidine) in the presence of protease inhibitors and 300 M PMSF.…”