Allosteric modulation of adenosine A1 and cannabinoid 1 receptor signaling by G‐peptides

Abstract

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 (A 1 R) and cannabinoid receptor 1 (CB 1 ). We expressed A 1 R and CB 1 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 N 6 ‐Cyclopentyladenosine (CPA) and 5’‐ N ‐Ethylcarboxamidoadenosine (NECA) for A 1 R and 2‐Arachidonoylglycerol (2‐AG) and WIN 55,212‐2 mesylate (WN) for CB 1 . 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 IP 1 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.

β2-adrenoceptor ligand efficacy is tuned by a two-stage interaction with the Gαs C terminus

“…While GPCR SPASM sensors have been repeatedly used to probe the effects of full agonists (14)(15)(16)(17)(18), their narrow dynamic range, combined with the sensitivity of sensor measurements to live-cell expression levels, have limited reliable measurements with partial agonists. Here, we overcome these limitations by vesiculating GPCR SPASM sensors into giant plasma membrane vesicles (GPMVs) and use this system to probe into the structural and dynamic basis of agonist efficacy in GPCRs.…”