Calcium-dependent dephosphorylation mediates the hyperosmotic and lysophosphatidic acid-dependent inhibition of natriuretic peptide receptor-B/guanylyl cyclase-B.
C-type natriuretic peptide binding to natriuretic peptide receptor-B (NPR-B) stimulates cGMP synthesis, which regulates vasorelaxation, cell proliferation, and bone growth. Here, we investigated the mechanistic basis for hyperosmotic and lysophosphatidic acid-dependent inhibition of NPR-B. Whole cell cGMP measurements and guanylyl cyclase assays indicated that acute hyperosmolarity decreased NPR-B activity in a reversible, concentration- and time-dependent manner, whereas chronic exposure had no effect. Acute hyperosmolarity elevated intracellular calcium in a concentration-dependent fashion that paralleled NPR-B desensitization. A calcium chelator, but not a protein kinase C inhibitor, blocked both calcium elevations and desensitization. Hyperosmotic medium stimulated NPR-B dephosphorylation, and the receptor was rapidly rephosphorylated and resensitized when the hypertonic media was removed. Lysophosphatidic acid also inhibited NPR-B in a calcium- and phosphorylation-dependent process, consistent with calcium being a universal regulator of NPR-B. The absolute requirement of dephosphorylation in this process was demonstrated by showing that a receptor with glutamates substituted at all known NPR-B phosphorylation sites is unresponsive to hyperosmotic stimuli. This is the first study to measure the phosphorylation state of an endogenous guanylyl cyclase and to link intracellular calcium elevations with its dephosphorylation.
Potthast
Regine
R
Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, 55405, USA.
Abbey-Hosch
Sarah E
SE
Antos
Laura K
LK
Marchant
Jonathan S
JS
Kuhn
Michaela
M
Potter
Lincoln R
LR
D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry* / Q000378:metabolism; D002460:Cell Line; D049109:Cell Proliferation; D002478:Cells, Cultured; D006152:Cyclic GMP / Q000737:chemistry / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D006162:Guanylate Cyclase / Q000737:chemistry* / Q000502:physiology*; D006801:Humans; D047468:Immunoprecipitation; D008246:Lysophospholipids / Q000737:chemistry*; D051379:Mice; D018613:Microscopy, Confocal; D041681:NIH 3T3 Cells; D009995:Osmosis; D010766:Phosphorylation; D051381:Rats; D017461:Receptors, Atrial Natriuretic Factor / Q000737:chemistry* / Q000502:physiology*; D018000:Receptors, Peptide / Q000737:chemistry; D012965:Sodium Chloride / Q000494:pharmacology; D013997:Time Factors; D014162:Transfection; D000099242:Atrial Natriuretic Factor Receptor B
D002118:Calcium; D006152:Cyclic GMP; D006162:Guanylate Cyclase; D008246:Lysophospholipids; D017461:Receptors, Atrial Natriuretic Factor; D018000:Receptors, Peptide; D012965:Sodium Chloride; D000099242:Atrial Natriuretic Factor Receptor B; C032881:lysophosphatidic acid