Promotion of G alpha i3 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 approximately 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 G alpha i3 in HEK293 cells and reduces the half-life of overexpressed G alpha i3-YFP. Thus, our findings suggest that GIPN is involved in the degradation of G alpha i3 subunits via the proteasome pathway. RGS-GAIP functions as a bifunctional adaptor that binds to G alpha subunits through its RGS domain and to GIPN through its cysteine string motif.
Fischer
Thierry
T
Department of Cellular and Molecular Medicine, University of California at San Diego, La Jolla, CA 92093, USA.
De Vries
Luc
L
Meerloo
Timo
T
Farquhar
Marilyn Gist
MG
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002896:Chromosomes, Human, Pair 6 / Q000235:genetics; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D020690:GTPase-Activating Proteins / Q000378:metabolism*; D005786:Gene Expression Regulation; D015723:Gene Library; D020962:Heterotrimeric GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D008025:Ligases / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D051379:Mice; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000378:metabolism*; D010902:Pituitary Gland / Q000166:cytology; D046988:Proteasome Endopeptidase Complex; D011485:Protein Binding; D025941:Protein Interaction Mapping; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D020710:RGS Proteins; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000502:physiology; D016415:Sequence Alignment; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D014162:Transfection; D020798:Two-Hybrid System Techniques; D025801:Ubiquitin / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases
D018076:DNA, Complementary; D020690:GTPase-Activating Proteins; D009097:Multienzyme Complexes; D020710:RGS Proteins; D011993:Recombinant Fusion Proteins; D025801:Ubiquitin; C097187:regulator of G-protein signalling 19; D044767:Ubiquitin-Protein Ligases; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; C498819:GNAI3 protein, human; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; C417708:Gnai3 protein, mouse; C498820:Gnai3 protein, rat; D020962:Heterotrimeric GTP-Binding Proteins; D008025:Ligases; C476759:Ostm1 protein, rat