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Mol Cell Biol, April 1998, p. 2164-2172, Vol. 18, No. 4
Molecular Biology and Virology Laboratory,
The Salk Institute for Biological Studies, La Jolla, California
92037
Received 30 September 1997/Returned for modification 3 November
1997/Accepted 8 January 1998
Natriuretic peptide receptor A (NPR-A) is the biological receptor
for atrial natriuretic peptide (ANP). Activation of the NPR-A guanylyl cyclase requires ANP binding to the extracellular domain
and ATP binding to a putative site within its cytoplasmic region. The
allosteric interaction of ATP with the intracellular kinase homology
domain (KHD) is hypothesized to derepress the carboxyl-terminal
guanylyl cyclase catalytic domain, resulting in the synthesis of the
second messenger, cyclic GMP. Here, we show that phosphorylation of the
KHD is essential for receptor activation. Using a combination of
phosphopeptide mapping techniques, we have identified six residues
within the ATP-binding domain (S497, T500, S502, S506, S510, and T513)
which are phosphorylated when NPR-A is expressed in HEK 293 cells. Mutation of any one of these Ser or Thr residues
to Ala caused reductions in the receptor phosphorylation state,
the number and pattern of phosphopeptides observed in tryptic
maps, and ANP-dependent guanylyl cyclase activity. The reductions were
not explained by decreases in NPR-A protein levels, as indicated by
immunoblot analysis and determinations of cyclase activity in the
presence of detergent. Conversion of Ser-497 to Ala resulted in the
most dramatic decrease in cyclase activity (~20% of wild-type
activity), but conversion to an acidic residue (Glu), which mimics the
charge of the phosphoserine moiety, had no effect. Simultaneous
mutation of five of the phosphorylation sites to Ala resulted in a
dephosphorylated receptor which was unresponsive to hormone and had
potent dominant negative inhibitory activity. We conclude that
phosphorylation of the KHD is absolutely required for hormone-dependent
activation of NPR-A.
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Phosphorylation of the Kinase Homology Domain Is
Essential for Activation of the A-Type Natriuretic Peptide
Receptor
*
Corresponding author. Mailing address: The Salk
Institute for Biological Studies, Molecular Biology and Virology
Laboratory, 10010 North Torrey Pines Road, La Jolla, CA 92037. Phone:
(619) 453-4100, ext. 1613. Fax: (619) 457-4765. E-mail:
lpotter{at}aim.salk.edu.
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