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 approximately 4-kilobase (kb) mRNA enriched in rat brain, is a Galpha(i)/Galpha(t)-binding protein that competes with Gbetagamma for interaction with Galpha(GDP) and acts as a guanine nucleotide dissociation inhibitor for heterotrimeric G-proteins. An approximately 2-kb AGS3 mRNA (AGS3-SHORT) is enriched in rat and human heart. We characterized the heart-enriched mRNA, identified the encoded protein, and determined its ability to interact with and regulate the guanine nucleotide-binding properties of G-proteins. Screening of a rat heart cDNA library, 5'-rapid amplification of cDNA ends, and RNase protection assays identified two populations of cDNAs (1979 and 2134 nucleotides plus the polyadenylation site) that diverged from the larger 4-kb mRNA (AGS3-LONG) in the middle of the protein coding region. Transfection of COS-7 cells with AGS3-SHORT cDNAs resulted in the expression of a major immunoreactive AGS3 polypeptide (M(r) approximately 23,000) with a translational start site at Met(495) of AGS3-LONG. Immunoblots indicated the expression of the M(r) approximately 23,000 polypeptide in rat heart. Glutathione S-transferase-AGS3-SHORT selectively interacted with the GDP-bound versus guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS)-bound conformation of Galpha(i2) and inhibited GTPgammaS binding to Galpha(i2). Protein interaction assays with glutathione S-transferase-AGS3-SHORT and heart lysates indicated interaction of AGS3-SHORT with Galpha(i1/2) and Galpha(i3), but not Galpha(s) or Galpha(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 stabilize the GDP-bound conformation of Galpha(i), but which differ in their tissue distribution and trafficking within the cell.
Department of Pharmacology, Medical University of South Carolina, Charleston, South Carolina 29425, USA.
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Pizzinat
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Takesono
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Lanier
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000378:metabolism; D016466:CHO Cells; D019556:COS Cells; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D002522:Chlorocebus aethiops; D006224:Cricetinae; D017931:DNA Primers; D018076:DNA, Complementary; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000378:metabolism; D015723:Gene Library; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism; D006352:Heart Ventricles; D020962:Heterotrimeric GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D009206:Myocardium / Q000378:metabolism*; D009928:Organ Specificity; D011487:Protein Conformation; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017384:Sequence Deletion; D014162:Transfection
D002352:Carrier Proteins; D017931:DNA Primers; D018076:DNA, Complementary; C498913:Gpsm1 protein, rat; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D006153:Guanosine Diphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D005982:Glutathione Transferase; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D019205:GTP-Binding Protein alpha Subunits, Gs; D020962:Heterotrimeric GTP-Binding Proteins