While allosteric modulation of GPCR signaling has gained prominence to address the need for receptor specificity, efforts have mainly focused on allosteric sites adjacent to the orthosteric ligand-binding pocket and lipophilic molecules that target transmembrane helices. In this study, we examined the allosteric influence of native peptides derived from the C-terminus of the Gα subunit (G-peptides) on signaling from two Gi-coupled receptors, adenosine A1 receptor (A1R) and cannabinoid receptor 1 (CB1). We expressed A1R and CB1 fusions with G-peptides derived from Gαs, Gαi, and Gαq in HEK 293 cells using systematic protein affinity strength modulation (SPASM) and monitored the impact on downstream signaling in the cell compared to a construct lacking G-peptides. We used agonists N6-Cyclopentyladenosine (CPA) and 5’-N-Ethylcarboxamidoadenosine (NECA) for A1R and 2-Arachidonoylglycerol (2-AG) and WIN 55,212-2 mesylate (WN) for CB1. G-peptides derived from Gαi and Gαq enhance agonist-dependent cAMP inhibition, demonstrating their effect as positive allosteric modulators of Gi-coupled signaling. In contrast, both G-peptides suppress agonist-dependent IP1 levels suggesting that they differentially function as negative allosteric modulators of Gq-coupled signaling. Taken together with our previous studies on Gs-coupled receptors, this study provides an extended model for the allosteric effects of G-peptides on GPCR signaling, and highlights their potential as probe molecules to enhance receptor specificity.
Bibliographical noteFunding Information:
This work was supported by the National Institutes of Health National Institute of General Medical Sciences [Grant 1R35GM126940‐01 to SS] and the National Heart Lung and Blood Institute [5F30HL146089‐02 to A.T]. We thank Ansley Semack for technical assistance. We also thank Michael Ritt for helpful discussions in writing this manuscript.
© 2020 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd, British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics.
- GTP-binding proteins
- SCH 442 416
- adenosine A1
- signal transduction