A constitutively "phosphorylated" guanylyl cyclase-linked atrial natriuretic peptide receptor mutant is resistant to desensitization

被引:61
作者
Potter, LR [1 ]
Hunter, T [1 ]
机构
[1] Salk Inst Biol Studies, Mol Biol & Virol Lab, La Jolla, CA 92037 USA
关键词
D O I
10.1091/mbc.10.6.1811
中图分类号
Q2 [细胞生物学];
学科分类号
071009 ; 090102 ;
摘要
Dephosphorylation of the natriuretic peptide receptor-ii (NPR-A) is hypothesized to mediate its desensitization in response to atrial natriuretic peptide (ANP) binding. Recently, we identified six phosphorylation sites within the kinase homology domain of NPR-A and determined that the conversion of these residues to alanine abolished the ability of the receptor to be phosphorylated or to be activated by ANP and ATP. In an attempt to generate a form of NPR-A that mimics a fully phosphorylated receptor but that is resistant to dephosphorylation, we engineered a receptor variant (NPR-A-6E) containing glutamate substitutions at all six phosphorylation sites. Consistent with the known ability of negatively charged glutamate residues to substitute functionally, in some cases, for phosphorylated residues, we found that NPR-A-6E was activated 10-fold by ANP and ATP. As determined by guanylyl cyclase assays, the hormone-stimulated activity of the wild-type receptor declined over time in membrane preparations in vitro, and this loss was blocked by the serine/threonine protein phosphatase inhibitor microcystin. In contrast, the activity of NPR-A-6E was more linear with time and was unaffected by microcystin. The nonhydrolyzable ATP analogue adenosine 5'-(beta,gamma-imino)triphosphate was half as effective as ATP in stimulating the wild-type receptor but was equally as potent in stimulating NPR-A-6E, suggesting that ATP is required to keep the wild-type but not 6E variant phosphorylated. Finally, the desensitization of NPR-A-6E in whole cells was markedly blunted compared with that of the wild-type receptor, consistent with its inability to shed the negative charge from its kinase homology domain via dephosphorylation. These data provide the first direct test of the requirement for dephosphorylation in guanylyl cyclase desensitization and they indicate that it is an essential component of this process.
引用
收藏
页码:1811 / 1820
页数:10
相关论文
共 33 条
[1]  
BENTLEY JK, 1986, J BIOL CHEM, V261, P4859
[2]  
CHINKERS M, 1991, ANNU REV BIOCHEM, V60, P553, DOI 10.1146/annurev.biochem.60.1.553
[3]   TARGETING OF A DISTINCTIVE PROTEIN-SERINE PHOSPHATASE TO THE PROTEIN KINASE-LIKE DOMAIN OF THE ATRIAL-NATRIURETIC-PEPTIDE RECEPTOR [J].
CHINKERS, M .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1994, 91 (23) :11075-11079
[4]   A MEMBRANE FORM OF GUANYLATE-CYCLASE IS AN ATRIAL NATRIURETIC PEPTIDE RECEPTOR [J].
CHINKERS, M ;
GARBERS, DL ;
CHANG, MS ;
LOWE, DG ;
CHIN, HM ;
GOEDDEL, DV ;
SCHULZ, S .
NATURE, 1989, 338 (6210) :78-83
[5]   THE PROTEIN-KINASE DOMAIN OF THE ANP RECEPTOR IS REQUIRED FOR SIGNALING [J].
CHINKERS, M ;
GARBERS, DL .
SCIENCE, 1989, 245 (4924) :1392-1394
[6]  
DOMINO SE, 1991, METHOD ENZYMOL, V195, P345
[7]   THE FAMILY OF GUANYLYL CYCLASE RECEPTORS AND THEIR LIGANDS [J].
DREWETT, JG ;
GARBERS, DL .
ENDOCRINE REVIEWS, 1994, 15 (02) :135-162
[8]   Dual role for adenine nucleotides in the regulation of the atrial natriuretic peptide receptor, guanylyl cyclase-A [J].
Foster, DC ;
Garbers, DL .
JOURNAL OF BIOLOGICAL CHEMISTRY, 1998, 273 (26) :16311-16318
[9]  
Furuya H, 1998, ZOOL SCI, V15, P507, DOI 10.2108/0289-0003(1998)15[507:MSAOPR]2.0.CO
[10]  
2