Advanced

Characterization of two recombinant PDE3 (cGMP-inhibited cyclic nucleotide phosphodiesterase) isoforms, RcGIP1 and HcGIP2, expressed in NIH 3006 murine fibroblasts and Sf9 insect cells

Leroy, Marie-Josephe; Degerman, Eva LU ; Taira, Masato; Murata, Taku; Wang, Lu Hua; Movsesian, Matthew A.; Meacci, Elisabetta and Manganiello, Vincent C. (1996) In Biochemistry 35(31). p.10194-10202
Abstract
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... (More)
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 (Km), 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. (Less)
Please use this url to cite or link to this publication:
author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Biochemistry
volume
35
issue
31
pages
10194 - 10202
publisher
The American Chemical Society
external identifiers
  • pmid:8756484
  • scopus:0029737541
ISSN
0006-2960
DOI
10.1021/bi952711t
language
English
LU publication?
yes
id
d5035849-53eb-499f-b078-27d8a19b9aa7 (old id 1110768)
date added to LUP
2008-07-25 14:45:46
date last changed
2017-02-12 03:33:19
@article{d5035849-53eb-499f-b078-27d8a19b9aa7,
  abstract     = {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 (Km), 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.},
  author       = {Leroy, Marie-Josephe and Degerman, Eva and Taira, Masato and Murata, Taku and Wang, Lu Hua and Movsesian, Matthew A. and Meacci, Elisabetta and Manganiello, Vincent C.},
  issn         = {0006-2960},
  language     = {eng},
  number       = {31},
  pages        = {10194--10202},
  publisher    = {The American Chemical Society},
  series       = {Biochemistry},
  title        = {Characterization of two recombinant PDE3 (cGMP-inhibited cyclic nucleotide phosphodiesterase) isoforms, RcGIP1 and HcGIP2, expressed in NIH 3006 murine fibroblasts and Sf9 insect cells},
  url          = {http://dx.doi.org/10.1021/bi952711t},
  volume       = {35},
  year         = {1996},
}