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PMID: 1379717 Published · ppublish English Comparative Study Journal Article Research Support, U.S. Gov't, P.H.S.

Cloning and expression of a Ca(2+)-inhibitable adenylyl cyclase from NCB-20 cells.

Proceedings of the National Academy of Sciences of the United States of America ·Vol. 89 ·No. 15 ·1992-08-01 ·Pages 6716-20

Yoshimura M, Cooper DM

Abstract

A cDNA that encodes an adenylyl cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] has been cloned from NCB-20 cells, in which adenylyl cyclase activity is inhibited by Ca2+ at physiological concentrations. The cDNA clone (5.8 kilobases) was isolated by polymerase chain reaction (PCR) using degenerate primers designed by comparison of three adenylyl cyclase sequences (types I, II, and III) and subsequent library screening. Northern analysis revealed expression of mRNA (6.1 kilobases) corresponding to this cDNA in cardiac tissue, which is a prominent source of Ca(2+)-inhibitable adenylyl cyclase. The clone encodes a protein of 1165 amino acids, whose hydrophilicity profile was very similar to those of other mammalian adenylyl cyclases that have recently been cloned. A noticeable difference between this protein and other adenylyl cyclases was a lengthy aminoterminal region before the first transmembrane span. Transient expression of this cDNA in the human embryonic kidney cell line 293 revealed a 3-fold increase in cAMP production in response to forskolin compared with control transfected cells. In purified plasma membranes from transfected cells, increased adenylyl cyclase activity was also detected, which was susceptible to inhibition by submicromolar Ca2+. Thus, this adenylyl cyclase seems to represent the Ca(2+)-inhibitable form that is encountered in NCB-20 cells, cardiac tissue, and elsewhere. Its identification should permit a determination of the structural features that determine the mode of regulation of adenylyl cyclase by Ca2+.

MeSH Terms
Adenylyl Cyclase Inhibitors Adenylyl Cyclases/genetics,metabolism Alprostadil/pharmacology Amino Acid Sequence Animals Base Sequence Calcium/pharmacology Cell Line Cell Membrane/enzymology Cloning, Molecular Colforsin/pharmacology Cyclic AMP/metabolism DNA/genetics,isolation & purification Guanylyl Imidodiphosphate/pharmacology Humans Hybrid Cells Kinetics Molecular Sequence Data Oligodeoxyribonucleotides Polymerase Chain Reaction Protein Conformation RNA/genetics,isolation & purification Sequence Homology, Nucleic Acid Transfection
Chemicals
Adenylyl Cyclase Inhibitors Oligodeoxyribonucleotides Colforsin Guanylyl Imidodiphosphate RNA DNA Cyclic AMP Adenylyl Cyclases Alprostadil Calcium
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Yoshimura M
Department of Pharmacology, University of Colorado Health Sciences Center, Denver 80262.
Cooper D M
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Article Info
Journal
Proceedings of the National Academy of Sciences of the United States of America
Abbr.
Proc Natl Acad Sci U S A
ISSN
0027-8424
DOI
10.1073/pnas.89.15.6716
Published
1992-08-01
Pages
6716-20
Language
English
Region
United States
NLM ID
7505876
PMCID
PMC49574
Subset
IM
Grants
NIGMS NIH HHS · GM32483 · United States
NINDS NIH HHS · NS28389 · United States
Databases
GENBANK
M93422
External Links
PubMed source DOI: 10.1073/pnas.89.15.6716 PMC Full Text View journal details
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