Biochemical and structural studies of signaling proteins have revealed critical features of peptide motifs at the interaction surfaces between proteins. Such information can be used to design small peptides that can be used as functional probes of specific interactions in signaling cascades. This article describes the use of a novel domain (the GoLoco motif) found in several members of the regulators of G-protein signaling (RGS) protein family to probe the specificity of Gα subunit involvement in the coupling of dopamine and somatostatin receptors to ion channels in the AtT20 neuroendocrine cell line. Peptides encoding the GoLoco motifs of RGS12 and AGS3 were perfused into single cells during electrical recording of agonist-induced current responses by whole cell patch clamp methods. The particular sequences chosen have been demonstrated to bind selectively to the GDP-bound form of Gαi, but not Gαo, and preclude association of Gβγ and Gαi subunits. A functional manifestation of this property is observed in the progressive uncoupling of D2 dopamine receptors and Kir3.13.2 channels with repeated agonist application. Similar uncoupling is not observed with somatostatin receptors nor with D2 receptors coupling to calcium channels, suggesting Gα subunit specificity in these signaling pathways. Motifs found in other proteins in the GPCR signaling machinery may also prove useful in assessing G-protein signaling specificity and complexity in single cells in the future.
ASJC Scopus subject areas