GoLoco motif peptides as probes of Galpha subunit specificity in coupling of G-protein-coupled receptors to ion channels.
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 Galpha 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 Galphai, but not Galphao, and preclude association of Gbetagamma and Galphai subunits. A functional manifestation of this property is observed in the progressive uncoupling of D2 dopamine receptors and Kir3.1/3.2 channels with repeated agonist application. Similar uncoupling is not observed with somatostatin receptors nor with D2 receptors coupling to calcium channels, suggesting Galpha 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.
Oxford
Gerry S
GS
Stark Neurosciences Research Institute, Indiana University School of Medicine, Indianapolis 46202, USA.
Webb
Christina K
CK
D000595:Amino Acid Sequence; D000818:Animals; D015220:Calcium Channels / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods; D002460:Cell Line; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels; D006801:Humans; D007473:Ion Channels / Q000378:metabolism*; D051379:Mice; D018408:Patch-Clamp Techniques; D010455:Peptides / Q000235:genetics / Q000378:metabolism*; D024661:Potassium Channels, Inwardly Rectifying / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D015290:Second Messenger Systems / Q000502:physiology
D015220:Calcium Channels; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels; D007473:Ion Channels; D010455:Peptides; D024661:Potassium Channels, Inwardly Rectifying; D043562:Receptors, G-Protein-Coupled