An isoform-specific interaction of the membrane anchors affects mammalian adenylyl cyclase type V activity

The nine membrane-bound mammalian adenylyl cyclases (ACs) contain two highl y diverged membrane anchors, M1 and M2, with six transmembrane spans each a nd two conserved cytosolic domains which coalesce into a pseudoheterodimeri c catalytic unit. Previously, the catalytic segments, bacterially expressed as soluble proteins, were characterized extensively whereas the function o f the membrane domains remained unexplored. Using the catalytic C1 and C2 d omains of AC type V we employed the membrane anchors from type V and VII AC s for construction of enzymes with duplicated, inverted, fully swapped and chimeric membrane anchors. Further, in the M1 membrane domain individual tr ansmembrane spans were removed or exchanged between type V and VII ACs. The constructs were expressed in HEK293 cells, the expression levels and membr ane localization was assessed by Western blotting. Cell-free basal, forskol in-, GTP gammaS-and G(s alpha)GTP gammaS-stimulated AC activities were dete rmined. The results demonstrate that enzymatic activities were only maintai ned when the M1 and M2 membrane domains were derived from either AC Vor VII . Constructs with chimeric membrane domains, i.e. M1 from type V and M2 fro m type VII AC or vice versa, were essentially inactive although the express ion levels and membrane localization appeared to be normal. The data indica te a functionally important interaction of the membrane domains of ACs in t hat they seem to interact in a pair-like, isoform delimited manner. This in teraction directly impinges on the formation of the catalytic interface. We propose that protein-protein interactions of the AC membrane domains may c onstitute another, yet unexplored level of AC regulation.