Splice variants of type VIII adenylyl cyclase. Differences in glycosylation and regulation by Ca2+/calmodulin.
Three alternatively spliced type VIII adenylyl cyclase messages have been identified by cDNA cloning and amplification from rat brain cDNA. Type VIII-A was previously referred to simply as type VIII (Cali, J. J., Zwaagstra, J. C., Mons, N., Cooper, D. M. F., and Krupinski, J. (1994) J. Biol. Chem. 269, 12190-12195). The types VIII-B and -C cDNAs differ from that of type VIII-A by deletion of 90 and 198 base pair exons, respectively, which encode a 30-amino acid extracellular domain with two consensus sites for N-linked glycosylation and a 66-amino acid cytoplasmic domain. Stable expression of types VIII-A, -B, and -C cDNAs in human embryonal kidney 293 (HEK-293) cells leads to the appearance of novel proteins, which are recognized by type VIII-specific antibodies and which co-migrate with immunoreactive species detected on immunoblots of rat brain membranes. Types VIII-A and -C are modified by N-linked glycosylation, while type VIII-B is insensitive to treatment with N-glycosidase F. An influx of extracellular Ca2+ stimulates cAMP accumulation in HEK-293 cells stably expressing type VIII-A, -B, or -C, but not in control cells. Adenylyl cyclase activity of each of the variants is stimulated by Ca2+/calmodulin and the EC50 for activation of type VIII-C is one fourth of that for either type VIII-A or -B. Type VIII-C also has a distinct Km for substrate, which is approximately 4-12-fold higher than that for types VIII-A or -B depending on whether Mn2+ or Mg2+ is the counterion for ATP. The differences in the structural and enzymatic properties of these three variants are discussed.
Cali
J J
JJ
Weis Center for Research, Geisinger Clinic, Danville, Pennsylvania 17822-2610, USA.
Parekh
R S
RS
Krupinski
J
J
D000262:Adenylyl Cyclases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017398:Alternative Splicing; D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000201:enzymology*; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D006031:Glycosylation; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D051381:Rats
D002147:Calmodulin; D018076:DNA, Complementary; D007527:Isoenzymes; D012333:RNA, Messenger; D000262:Adenylyl Cyclases; D002118:Calcium