Characterization of transmembrane domains of class IIIa adenylyl cyclase: A three state model

Abstract

cAMP uses a plethora of ways to be integrated into regulatory systems hence becoming a universal second messenger. Generation of such a crucial moiety is via a widespread signaling protein, adenylyl cyclase (AC). Classically, a membrane-bound AC has a N-terminal transmembrane domain (TM) and a transducer domain which are linked to the C-terminal catalytic domain. And, these features are dominantly displayed in pharmacologically most relevant species i.e. mammalian ACs. Mammalian ACs (mACs) have nine membrane-bound isoforms, with two dissimilar hexahelical membrane anchors that are isoform-specifically conserved for ›500 million years and which hold ≈40% protein. For which a physiological function is not known. So, a typical mode of regulation for this enzyme known is indirect i.e. via GPCRs (where G-protein  subunit, Gsα, is released and binds to the catalytic domain of an AC). Since, understanding the myriad aspects of cAMP signaling concerning the regulation of its enzyme, the role of the hexahelical membrane domains has remained enigmatic. Despite many speculations that were made regarding the role of these huge transmembrane helices no role was substantiated. A direct investigation for the regulatory function of these TMs was seemingly unapproachable as no ligand was known. Therefore, I exchanged both membrane anchors of the human adenylyl cyclase isoform 2 (hAC2) with the membrane domain of the quorum-sensing receptor from Vibrio harveyi, CqsS, for which the ligand is known, CAI-1. A functional chimera was attained, where cyclase activity was stimulated by Gsα but CAI-1 by itself had no effect. Here I showcase a new layer of regulation of adenylyl cyclase: where CAI-1 inhibited Gsα-stimulated cyclase activity thus shifting the concentration-response curve to the right and attenuating the maximal response. The results identify a potential regulation of mACs by the membrane anchors, as receptors, and Gsα; thus, making each mAC isoform approachable for the search of the ligands. Further bolstering the hypothesis, I report serum inhibiting AC activity in membranes isolated from rat brain cortex. Concomitantly, another cyclase stimulating factor, forskolin, was tested. Using hAC2 expressed in Sf9 cells a similar pattern of inhibition was observed in the presence of serum i.e. lowering stimulation by both, forskolin and forskolin + Gsα. Thus, marking the serum as a potential fluid to search for the ligands. Taken together, the data determine a putative pathway of signaling via AC membrane anchors as receptors to the catalytic domain. This resulted in a three-state model, defining a new orchestrated form of AC regulation and an approach to search for ligands, 31 years after its sequencing.