Gsα stimulation of mammalian adenylate cyclases regulated by their hexahelical membrane anchors

Abstract

Mammalian adenylate cyclases (ACs) are pseudoheterodimers with dissimilar hexahelical membrane-anchors, isoform-specifically conserved for more than half a billion years. We exchanged both membrane anchors of the AC isoform 2 by the isosteric quorum-sensing receptor from Vibrio, CqsS, which has a ligand, Cholera-Autoinducer-1 (CAI-1). In the chimera, AC activity was stimulated by Gsα, CAI-1 had no effect. Surprisingly, CAI-1 inhibited Gsα stimulation. We report that Gsα stimulation of human AC isoforms 2, 3, 5, and 9 expressed in Sf9 cells is inhibited by serum as is AC activity in membranes from rat brain cortex. AC2 activation by forskolin or forskolin/Gsα was similarly inhibited. Obviously, serum contains as yet unidentified factors affecting AC activity. The data establish a linkage in ACs, in which the membrane anchors, as receptors, transduce extracellular signals to the cytosolic catalytic dimer. A mechanistic three state model of AC regulation is presented compatible with all known regulatory inputs into mammalian ACs. The data allow designating the membrane anchors of mammalian ACs as orphan receptors and establish a new level of AC regulation. keywords: adenylate cyclases / cyclic AMP / membrane anchor / quorum-sensor / receptor

Structure ofMycobacterium tuberculosisCya, an evolutionary ancestor of the mammalian membrane adenylyl cyclases

“…It is clear that the HD region plays a vital part in AC and GC assembly and stability. This is evident from our experiments with the M. intracellulare Cya ( 17 ), as well as the results of others using Cya and mammalian ACs as model enzymes ( 10, 26, 28 ). It remains to be determined whether any agents can elicit conformational changes in the membrane domain of Cya (or in any of the mammalian membrane ACs), leading to substantial changes in the HD similar in scale to the changes observed in the sGC during its activation.…”

“…The two functions are not mutually exclusive, as the ligand interactions with the membrane region of the AC may influence the HD assembly and thus regulate the cyclase function. Previous work utilizing chimeric constructs composed of fragments of the quorum sensing receptor CqsS from Vibrio harveyi and Cya ( 20 ) or mammalian membrane ACs ( 28 ) revealed that the membrane anchors of the ACs may act as orphan receptors for yet unknown ligands. Together with the proposed functional coupling between the TM domain and the catalytic site of Cya, the structure described here is consistent with these findings, offering molecular insights into the potential receptor role of the membrane anchor of a model membrane AC.…”

Distinct glycerophospholipids potentiate Gsα-activated adenylyl cyclase activity

“…because we expected to characterize an inhibitory input (Graziano, Freissmuth et al, 1991, Seth et al, 2020. Most surprisingly, we discovered that 1-stearoyl-2-docosahexaenoyl-phosphatidic acid (SDPA) potentiated mAC3 up to 7-fold above the 16-fold activation already exerted by 600 nM Gsα alone (Fig.…”

“…We then carried out a lipidomic analysis with the pH 1 and the pH 6 fractions (Matyash, Liebisch et al, 2008, Vvedenskaya, Rose et al, 2021. Based on our previous data we expected ligands which inhibit Gsα-activated mAC activities (Seth et al, 2020). Therefore we concentrated on lipids prevalent in the pH 1 fraction.…”

“…it sequentially involves (i) the extracellular activation of G-protein-coupled receptors, (ii) intracellular release of the Gsα subunit from a trimeric G-protein and, (iii), as a last step mAC activation by the free αsubunit (Dessauer et al, 2017, Sadana & Dessauer, 2009. In contrast, we recently have assigned a direct regulatory role mediated by the membrane domains of the 9 mAC isoforms as receptors (Beltz, Bassler et al, 2016, Seth, Finkbeiner et al, 2020. In this proposal the mAC receptors are comprised of the two hexahelical domains each connected to a cytosolic catalytic domain, C1 and C2, via highly conserved cyclase-transducing-elements (Dessauer et al, 2017, Seth et al, 2020, Ziegler, Bassler et al, 2017.…”