ACTIVATOR OF G‐PROTEIN SIGNALING 3: THE ROLE OF THE TETRATRICOPEPTIDE REPEAT DOMAIN IN SUBCELLULAR POSITIONING OF THE PROTEIN

Abstract

AGS3 is a receptor independent activator of G‐protein signaling involved in unexpected functional diversity for G‐protein signaling systems. AGS3 has 7 tetratricopeptide (TPR) motifs upstream of 4 G‐protein regulatory (GPR) motifs, each of which bind and stabilize the GDP bound conformation of Giα. The TPR domain is postulated to play an important regulatory role in subcellular positioning of AGS3 and influence its interaction with G‐protein. We asked which regions of the TPR domain determine the subcellular distribution of AGS3. Disruption of the TPR organizational structure by deletion or point mutations leads to a redistribution of AGS3 to punctate structures throughout the cytoplasm similar to pre‐aggresomal assemblies. This re‐direction of AGS3 within the cell is also observed with a single nucleotide polymorphism (SNP) that alters codon usage within the TPR domain. The cellular distribution of the AGS3‐SNP to the punctate structures is rescued by increased expression of Giα3 but not a constitutively active mutant of Giα3. BRET analysis of the interaction of AGS3 with Giα3 indicated that AGS3‐SNP exhibited increased net BRET as compared to wild type AGS3. These data indicate intramolecular communication between the TPR and GPR domains of AGS3 as an important determinant of the positioning of the protein within the cell and its interaction with G‐protein.