TY - JOUR
T1 - Characterization of two recombinant PDE3 (cGMP-inhibited cyclic nucleotide phosphodiesterase) isoforms, RcGIP1 and HcGIP2, expressed in NIH 3006 murine fibroblasts and Sf9 insect cells
AU - Leroy, Marie Josephe
AU - Degerman, Eva
AU - Taira, Masato
AU - Murata, Taku
AU - Wang, Lu Hua
AU - Movsesian, Matthew A.
AU - Meacci, Elisabetta
AU - Manganiello, Vincent C.
N1 - Copyright:
Copyright 2007 Elsevier B.V., All rights reserved.
PY - 1996
Y1 - 1996
N2 - cDNAs encoding PDE3 [cGMP-inhibited cyclic nucleotide phosphodiesterase (cGI PDE)] isoforms, cGIP1 and cGIP2, have been cloned from rat (R) and human (H) cDNA libraries. The deduced amino acid sequences of RcGIP1 and HcGIP2 are very similar in their conserved catalytic domains but differ in their N- terminal regulatory domains [Meacci, E., et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 3721-3725; Taira, M., et al. (1993) J. Biol. Chem. 268, 18573- 18579]. cDNAs encoding both rat adipocyte RcGIP1 and human myocardial HcGIP2 (full-length forms and truncated forms lacking much of the putative N- terminal domain) were expressed in NIH 3006 fibroblasts and in Sf9 insect cells. The recombinant proteins exhibited the expected subunit molecular mass, immunologic reactivities, and characteristics of native membrane- associated forms of the enzymes, e.g., high affinity for cAMP (K(m)), sensitivity to the selective cGI PDE inhibitors OPC 3689 and OPC 3911 and to cGMP. The full-length recombinants were predominantly particulate, whereas the truncated HcGIP2 forms were cytosolic suggesting that N-terminal domains contain structural determinants important for membrane association. Both fibroblast RcGIP1 and authentic adipocyte cGI PDE were phosphorylated in vitro by cAMP-dependent protein kinase; tryptic [32P]peptides released from rat adipocyte 32P-cGI PDE and 32P-RcGIP1 exhibited identical electrophoretic profiles suggesting that the same peptides are phosphorylated in both.
AB - cDNAs encoding PDE3 [cGMP-inhibited cyclic nucleotide phosphodiesterase (cGI PDE)] isoforms, cGIP1 and cGIP2, have been cloned from rat (R) and human (H) cDNA libraries. The deduced amino acid sequences of RcGIP1 and HcGIP2 are very similar in their conserved catalytic domains but differ in their N- terminal regulatory domains [Meacci, E., et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 3721-3725; Taira, M., et al. (1993) J. Biol. Chem. 268, 18573- 18579]. cDNAs encoding both rat adipocyte RcGIP1 and human myocardial HcGIP2 (full-length forms and truncated forms lacking much of the putative N- terminal domain) were expressed in NIH 3006 fibroblasts and in Sf9 insect cells. The recombinant proteins exhibited the expected subunit molecular mass, immunologic reactivities, and characteristics of native membrane- associated forms of the enzymes, e.g., high affinity for cAMP (K(m)), sensitivity to the selective cGI PDE inhibitors OPC 3689 and OPC 3911 and to cGMP. The full-length recombinants were predominantly particulate, whereas the truncated HcGIP2 forms were cytosolic suggesting that N-terminal domains contain structural determinants important for membrane association. Both fibroblast RcGIP1 and authentic adipocyte cGI PDE were phosphorylated in vitro by cAMP-dependent protein kinase; tryptic [32P]peptides released from rat adipocyte 32P-cGI PDE and 32P-RcGIP1 exhibited identical electrophoretic profiles suggesting that the same peptides are phosphorylated in both.
UR - http://www.scopus.com/inward/record.url?scp=0029737541&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0029737541&partnerID=8YFLogxK
U2 - 10.1021/bi952711t
DO - 10.1021/bi952711t
M3 - Article
C2 - 8756484
AN - SCOPUS:0029737541
SN - 0006-2960
VL - 35
SP - 10194
EP - 10202
JO - Biochemistry
JF - Biochemistry
IS - 31
ER -