Thierry Fischer

Regulators of G Protein Signaling Proteins as Determinants of the Rate of Desensitization of Presynaptic Calcium Channels

Norepinephrine inhibits ω-conotoxin GVIA-sensitive presynaptic Ca2+ channels in chick dorsal root ganglion neurons through two pathways, one mediated by Go and the other by Gi. These pathways desensitize at different rates. We have found that recombinant Gα interacting protein (GAIP) and regulators of G protein signaling (RGS)4 selectively accelerate the rate of desensitization of Go- and Gi-mediated pathways, respectively. Blockade of endogenous RGS proteins using antibodies raised against Gα interacting protein and RGS4 slows the rate of desensitization of these pathways in a selective manner. These results demonstrate that different RGS proteins may interact with Gi and Go selectively, giving rise to distinct time courses of transmitter-mediated effects.

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

We have isolated an RGS-GAIP interacting protein that links RGS proteins to protein degradation. GIPN (GAIP interacting protein N terminus) is a 38-kDa protein with an N-terminal leucine-rich region, a central RING finger-like domain, and a putative C-terminal transmembrane domain. GIPN binds exclusively to RGS proteins of subfamily A, RGS-GAIP, RGSZ1, and RGSZ2. The N-terminal leucine-rich region of GIPN interacts with the cysteine-rich motif of RGS-GAIP. GIPN mRNA is ubiquitously expressed, and GIPN is found on the plasma membrane of transfected HEK293 cells. Endogenous GIPN is concentrated along the basolateral plasma membrane of proximal and distal tubules in rat kidney, where many G protein-coupled receptors and some G proteins are also located. Two immunoreactive species are found in rat kidney, a 38-kDa cytosolic form and an ≈94-kDa membrane form. GIPN shows Zn2+- and E1/E2-dependent autoubiquitination in vitro, suggesting that it has E3 ubiquitin ligase activity. Overexpression of GIPN stimulates proteasome-dependent reduction of endogenous Gαi3 in HEK293 cells and reduces the half-life of overexpressed Gαi3-YFP. Thus, our findings suggest that GIPN is involved in the degradation of Gαi3 subunits via the proteasome pathway. RGS-GAIP functions as a bifunctional adaptor that binds to Gα subunits through its RGS domain and to GIPN through its cysteine string motif.