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 quorum-sensing receptor from Vibrio harveyi, 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 isolated 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.,Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.