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

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.

In search of a function for the membrane anchors of class IIIa adenylate cyclases.

Nine pseudoheterodimeric mammalian adenylate cyclases possess two dissimilar hexahelical membrane domains (TM1 and TM2), two dissimilar cyclase-transducing-elements (CTEs) and two complementary catalytic domains forming a catalytic dimer (often termed cyclase-homology-domain, CHD). Canonically, these cyclases are regulated by G-proteins which are released upon ligand activation of G-protein-coupled receptors. So far, a biochemical function of the membrane domains beyond anchoring has not been established. For almost 30 years, work in our laboratory was based on the hypothesis that these voluminous membrane domains possess an additional physiological, possibly regulatory function. Over the years, we have generated numerous artificial fusion proteins between the catalytic domains of various bacterial adenylate cyclases which are active as homodimers and the membrane receptor domains of known bacterial signaling proteins such as chemotaxis receptors and quorum-sensors which have known ligands. Here we summarize the current status of our experimental efforts. Taken together, the data allow the conclusion that the hexahelical mammalian membrane anchors as well as similar membrane anchors from bacterial adenylate cyclase congeners are orphan receptors. A search for as yet unknown ligands of membrane-delimited adenylate cyclases is now warranted.

CyaC, a redox-regulated adenylate cyclase of Sinorhizobium meliloti with a quinone responsive diheme-B membrane anchor domain.

The nucleotide cyclase CyaC of Sinorhizobium meliloti is a member of class III adenylate cyclases (AC), a diverse group present in all forms of life. CyaC is membrane-integral by a hexahelical membrane domain (6TM) with the basic topology of mammalian ACs. The 6TM domain of CyaC contains a tetra-histidine signature that is universally present in the membrane anchors of bacterial diheme-B succinate-quinone oxidoreductases. Heterologous expression of cyaC imparted activity for cAMP formation from ATP to Escherichia coli, whereas guanylate cyclase activity was not detectable. Detergent solubilized and purified CyaC was a diheme-B protein and carried a binuclear iron-sulfur cluster. Single point mutations in the signature histidine residues caused loss of heme-B in the membrane and loss of AC activity. Heme-B of purified CyaC could be oxidized or reduced by ubiquinone analogs (Q0 or Q0 H2 ). The activity of CyaC in bacterial membranes responded to oxidation or reduction by Q0 and O2 , or NADH and Q0 H2 respectively. We conclude that CyaC-like membrane anchors of bacterial ACs can serve as the input site for chemical stimuli which are translated by the AC into an intracellular second messenger response.