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 highly diverged membrane anchors, M1 and M2, with six transmembrane spans each and two conserved cytosolic domains which coalesce into a pseudoheterodimeric catalytic unit. Previously, the catalytic segments, bacterially expressed as soluble proteins, were characterized extensively whereas the function of the membrane domains remained unexplored. Using the catalytic C1 and C2 domains of AC type V we employed the membrane anchors from type V and VII ACs for construction of enzymes with duplicated, inverted, fully swapped and chimeric membrane anchors. Further, in the M1 membrane domain individual transmembrane spans were removed or exchanged between type V and VII ACs. The constructs were expressed in HEK293 cells, the expression levels and membrane localization was assessed by Western blotting. Cell-free basal, forskolin-, GTP gamma S-and G(s alpha)/GTP gamma S-stimulated AC activities were determined. The results demonstrate that enzymatic activities were only maintained when the M1 and M2 membrane domains were derived from either AC V or VII. Constructs with chimeric membrane domains, i.e. M1 from type V and M2 from type VII AC or vice versa, were essentially inactive although the expression levels and membrane localization appeared to be normal. The data indicate a functionally important interaction of the membrane domains of ACs in that they seem to interact in a pair-like, isoform delimited manner. This interaction directly impinges on the formation of the catalytic interface. We propose that protein-protein interactions of the AC membrane domains may constitute another, yet unexplored level of AC regulation.
Seebacher
T
T
Pharmaceutical Biochemistry, Institute of Pharmacy, University of Tübingen, Germany.
Linder
J U
JU
Schultz
J E
JE
D000262:Adenylyl Cyclases / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D011817:Rabbits; D011994:Recombinant Proteins / Q000378:metabolism
D007527:Isoenzymes; D011994:Recombinant Proteins; D005576:Colforsin; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases; C419510:adenylyl cyclase type V