Identification of a truncated form of the G-protein regulator AGS3 in heart that lacks the tetratricopeptide repeat domains

AGS3, a 650-amino acid protein encoded by an similar to4-kilobase (kb) mRNA enriched in rat brain, is a G alpha (i)/G alpha (t)-binding protein that c ompetes with G beta gamma for interaction with G alpha (GDP) and acts as a guanine nucleotide dissociation inhibitor for heterotrimeric G-proteins, An similar to2-kb AGS3 mRNA (AGS3-SHORT) is enriched in rat and human heart. We characterized the heart-enriched mRNA, identified the encoded protein, a nd determined its ability to interact with and regulate the guanine nucleot ide-binding properties of G-proteins. Screening of a rat heart cDNA library , 5 ' -rapid amplification of cDNA ends, and RNase protection assays identi fied two populations of cDNAs (1979 and 2134 nucleotides plus the polyadeny lation site) that diverged from the larger 4-kb mRNA (AGS3-LONG) in the mid dle of the protein coding region. Transfection of COS-7 cells with AGS3-SHO RT cDNAs resulted in the expression of a major immunoreactive AGS3 polypept ide (M-r similar to 23,000) with a translational start site at Met(495) of AGS3-LONG. Immunoblots indicated the expression of the M-r similar to 23,00 0 polypeptide in rat heart. Glutathione S-transferase-AGS3-SHORT selectivel y interacted with the GDP-bound versus guanosine 5 ' -O-(3-thiotriphosphate ) (GTP gammaS)-bound conformation of G alpha (i2) and inhibited GTP gammaS binding to G alpha (i2). Protein interaction assays with glutathione S-tran sferase-AGS3-SHORT and heart ly; sates indicated interaction of AGS3-SHORT with G alpha (i1/2), and G alpha (i3), but not G alpha (s) or G alpha (q). Immunofluorescent imaging and subcellular fractionation following transient expression of AGS3-SHORT and AGS3-LONG in COS-7 and Chinese hamster ovary cells indicated distinct subcellular distributions of the two forms of AGS3 , Thus, AGS3 exists as a short and long form, both of which apparently stab ilize the GDP-bound conformation of G alpha (i), but which differ in their tissue distribution and trafficking within the cell.