One of the key questions for Activator of G‐protein Signaling 3 (AGS3) and related proteins containing G‐protein regulatory (GPR) motifs is what controls their positioning within the cell and their interaction with G‐proteins. AGS3 has 7 tetratricopeptide (TPR) motifs upstream of 4 GPR motifs, each of which bind and stabilize the GDP bound conformation of Gialpha. The TPR domain of AGS3 serves as a key determinant of subcellular positioning through its interaction with specific binding proteins. We addressed the role of individual TPR domains and these binding partners in regulating the interaction of AGS3 with G‐protein in living cells (HEK‐293) by bioluminescence resonance energy transfer (BRET) with the donor‐acceptor pair AGS3‐ Renilla luciferase (RLuc) and Gialpha1‐YFP. Progressive deletion of TPR domains increased net BRET compared to wild type AGS3. Similar increase in net BRET signal occurred upon disruption of the TPR domain such as a single nucleotide polymorphism identified between the 4th and the 5th TPR motif. Co‐transfection of AGS3‐binding partners LKB1, Inscuteable or Frmpd1, which bind to the TPR or linker domains in AGS3, with AGS3‐RLuc and Gialpha1‐YFP differentially regulated the interaction of the two proteins. These data indicate that the TPR domain regulates the interaction of the AGS3 GPR domain with G‐proteins.