Critical cysteines in the functional interaction of adenylyl cyclase isoform 6 with Gαs

Anjali Y Bhagirath; Vikram Bhatia; Manoj Reddy Medapati; Nisha Singh; Martha Hinton; Prashen Chelikani; Shyamala Dakshinamurti

doi:10.1096/fba.2021-00073

Critical cysteines in the functional interaction of adenylyl cyclase isoform 6 with Gαs

FASEB Bioadv. 2021 Nov 22;4(3):180-196. doi: 10.1096/fba.2021-00073. eCollection 2022 Mar.

Authors

Anjali Y Bhagirath^{1

2}, Vikram Bhatia^{1

2}, Manoj Reddy Medapati², Nisha Singh², Martha Hinton^{1

3}, Prashen Chelikani^{1

2}, Shyamala Dakshinamurti^{1

3

4}

Affiliations

¹ Biology of Breathing Theme Children's Hospital Research Institute of Manitoba (CHRIM) Winnipeg Manitoba Canada.
² Department of Oral Biology University of Manitoba Winnipeg Manitoba Canada.
³ Department of Physiology and Pathophysiology University of Manitoba Winnipeg Manitoba Canada.
⁴ Department of Pediatrics University of Manitoba Winnipeg Manitoba Canada.

Abstract

Activation of adenylyl cyclases (ACs) by G-protein Gαs catalyzes the production of cyclic adenosine monophosphate (cAMP), a key second messenger that regulates diverse physiological responses. There are 10 AC isoforms present in humans, with AC5 and AC6 proposed to play vital roles in cardiac function. We have previously shown that under hypoxic conditions, AC6 is amenable to post-translational modification by nitrosylation, resulting in decreased AC catalytic activity. Using a computational model of the AC6-Gαs complex, we predicted key nitrosylation-amenable cysteine residues involved in the interaction of AC6 with Gαs and pursued a structure-function analysis of these cysteine residues in both AC6 and Gαs. Our results based on site-directed mutagenesis of AC6 and Gαs, a constitutively active Gαs, AC activity, and live cell intracellular cAMP assays suggest that Cys1004 in AC6 (subunit C2) and Cys237 in Gαs are present at the AC-Gαs interface and are important for the activation of AC6 by Gαs. We further provide mechanistic evidence to show that mutating Cys 1004 in the second catalytic domain of AC6 makes it amenable to inhibition by Gαi, which may account for decreased functional activity of AC6 when this residue is unavailable.

Keywords: G proteins; adenylyl cyclase; cyclic AMP; cysteine; mutational analysis.