In:
Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 95, No. 4 ( 1998-02-17), p. 1466-1471
Abstract:
A recently cloned isoform of cGMP-dependent protein kinase (cGK), designated type II, was implicated as the mediator of cGMP-provoked intestinal Cl − secretion based on its localization in the apical membrane of enterocytes and on its capacity to activate cystic fibrosis transmembrane conductance regulator (CFTR) Cl − channels. In contrast, the soluble type I cGK was unable to activate CFTR in intact cells, although both cGK I and cGK II could phosphorylate CFTR in vitro . To investigate the molecular basis for the cGK II isotype specificity of CFTR channel gating, we expressed cGK II or cGK I mutants possessing different membrane binding properties by using adenoviral vectors in a CFTR-transfected intestinal cell line, and we examined the ability of cGMP to phosphorylate and activate the Cl − channel. Mutation of the cGK II N-terminal myristoylation site (Gly 2 → Ala) reduced cGK II membrane binding and severely impaired cGK II activation of CFTR. Conversely, a chimeric protein, in which the N-terminal membrane-anchoring domain of cGK II was fused to the N terminus of cGK Iβ, acquired the ability to associate with the membrane and activate the CFTR Cl − channel. The potency order of cGK constructs for activation of CFTR (cGK II 〉 membrane-bound cGK I chimer ≫ nonmyristoylated cGK II 〉 cGK Iβ) correlated with the extent of 32 P incorporation into CFTR observed in parallel measurements. These results strongly support the concept that membrane targeting of cGK is a major determinant of CFTR Cl − channel activation in intact cells.
Type of Medium:
Online Resource
ISSN:
0027-8424
,
1091-6490
DOI:
10.1073/pnas.95.4.1466
Language:
English
Publisher:
Proceedings of the National Academy of Sciences
Publication Date:
1998
detail.hit.zdb_id:
209104-5
detail.hit.zdb_id:
1461794-8
SSG:
11
SSG:
12
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