Molecular cloning of the rat adipocyte hormone-sensitive cyclic GMP-inhibited cyclic nucleotide phosphodiesterase.
Two distinct but related cGMP-inhibited cyclic nucleotide phosphodiesterase (cGI PDE) cDNAs were cloned from rat adipose tissue cDNA libraries. The open reading frame (3324 base pairs) of RcGIP1 encodes 1108 amino acids, including a hydrophobic membrane-associated domain in the NH2-terminal portion and, in the COOH-terminal portion, a putative catalytic domain conserved among all mammalian PDEs which is preceded by a putative regulatory domain that contains three consensus cAMP-dependent protein kinase phosphorylation sites and followed by a hydrophilic COOH-terminal domain. The carboxyl-terminal portion including the conserved domain was expressed as a glutathione S-transferase fusion protein and exhibited cAMP PDE activity which was inhibited by cilostamide, a specific cGI PDE inhibitor. RcGIP1 cDNA hybridizes strongly with RNA from isolated adipocytes, and its mRNA increases dramatically during differentiation of 3T3-L1 adipocytes. The deduced sequence of the second partial cDNA clone (RcGIP2 clone 53B) is highly homologous to the corresponding region of human cardiac cGI PDE cDNA. RcGIP2 cDNA hybridized strongly with rat cardiac tissue RNA and weakly if at all with RNA from rat adipocytes or 3T3-L1 fibroblasts or adipocytes. We suggest that RcGIP1 represents the hormone-sensitive, membrane-associated rat adipocyte cGI PDE and RcGIP2, a cGI PDE from vascular elements in rat adipose tissue.
Laboratory of Cellular Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892.
M
M
Taira
S C
SC
Hockman
J C
JC
Calvo
M
M
Taira
P
P
Belfrage
V C
VC
Manganiello
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics*; D000273:Adipose Tissue / Q000201:enzymology*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001792:Blood Platelets / Q000201:enzymology; D003001:Cloning, Molecular; D016384:Consensus Sequence; D006152:Cyclic GMP / Q000494:pharmacology*; D004247:DNA / Q000737:chemistry / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D010766:Phosphorylation; D015363:Quinolones / Q000494:pharmacology; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D012045:Regulatory Sequences, Nucleic Acid; D017385:Sequence Homology
D015363:Quinolones; D012333:RNA, Messenger; C021294:cilostamide; D004247:DNA; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D006152:Cyclic GMP