Regulation of forskolin interactions with type I, II, V, and VI adenylyl cyclases by Gs alpha.
Several forms of adenylyl cyclase (types I, II, V, and VI) have been expressed using the recombinant baculovirus expression system in Sf9 cells. The activation of type I adenylyl cyclase by forskolin and Gs alpha was not greater than additive. In contrast, there was synergistic activation of type II, V, and VI adenylyl cyclases by Gs alpha and forskolin. Gs alpha potentiated the effect of forskolin on type II adenylyl cyclase to the greatest extent. Type I and II adenylyl cyclases were photolabeled specifically by an iodinated photoaffinity derivative of forskolin ([125I]-6-AIPP-Fsk). Type I adenylyl cyclase was photolabeled efficiently in the absence of Gs alpha, and the addition of Gs alpha only slightly increased the labeling efficiency. In contrast, type II adenylyl cyclase was not photolabeled efficiently in the absence of Gs alpha, and the addition of Gs alpha greatly enhanced the labeling efficiency. Photolabeling of type V and VI adenylyl cyclases was detected only in the presence of Gs alpha. Neither calcium/calmodulin nor G protein beta gamma subunits modulated the photolabeling of type I or II adenylyl cyclases. Another iodinated derivative of forskolin, [125I]-6-IHPP-fsk, bound to Sf9 cell membranes expressing type I adenylyl cyclase with high affinity in a filtration binding assay, and the specific binding was not enhanced by the addition of Gs alpha. In contrast, specific binding of [125I]-6-IHPP-Fsk to membranes expressing type II adenylyl cyclase was detected only in the presence of Gs alpha.(ABSTRACT TRUNCATED AT 250 WORDS)
Sutkowski
E M
EM
Center for Biologics Evaluation and Research, Food and Drug Administration, Rockville, Maryland 20852.
Tang
W J
WJ
Broome
C W
CW
Robbins
J D
JD
Seamon
K B
KB
D000262:Adenylyl Cyclases / Q000235:genetics / Q000378:metabolism*; D000345:Affinity Labels; D000818:Animals; D001386:Azides / Q000494:pharmacology; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D005576:Colforsin / Q000031:analogs & derivatives / Q000494:pharmacology*; D004224:Diterpenes; D004357:Drug Synergism; D004789:Enzyme Activation / Q000187:drug effects; D019204:GTP-Binding Proteins / Q000502:physiology*; D015870:Gene Expression; D007457:Iodine Radioisotopes; D010777:Photochemistry; D011994:Recombinant Proteins / Q000378:metabolism; D018411:Spodoptera / Q000201:enzymology
D000345:Affinity Labels; D001386:Azides; D004224:Diterpenes; D007457:Iodine Radioisotopes; D011994:Recombinant Proteins; C069863:N-(3-(4-azido-3-iodophenyl)propionamide)-6-aminoethylcarbamylforskolin; D005576:Colforsin; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases