Protein kinase C-dependent desensitization of the atrial natriuretic peptide receptor is mediated by dephosphorylation.
Stimulation of guanylyl cyclase A (GC-A) by atrial natriuretic peptide (ANP) is antagonized by activators of protein kinase C (PKC). Thus, it has been suggested that PKC phosphorylates and desensitizes GC-A. Here, we have developed stable GC-A transfectants of NIH3T3 cells, which display marked reductions in hormone-dependent cGMP elevations and guanylyl cyclase activity after incubation with ANP or phorbol 12-myristate 13-acetate (PMA). ANP binding and immunoblot analysis indicated that the decreases were not due to receptor internalization or degradation. GC-A isolated from 32PO4-labeled cells contained phosphoserine and phosphothreonine. ANP and/or PMA addition caused substantial decreases in the 32P content of the receptor that coincided with reductions in hormone-dependent guanylyl cyclase activity. The specific PKC inhibitor, GF-109203X, completely blocked the PMA-dependent dephosphorylation and desensitization of GC-A but failed to inhibit either ANP-dependent process. Tryptic phosphopeptide maps of GC-A isolated from ANP- or PMA-treated cells were unique, suggesting that the sites that dephosphorylated in response to each agent were different. In contrast to previous reports, we conclude that PMA and ANP desensitization of GC-A are distinct events mediated by dephosphorylation of specific residues through PKC-dependent and -independent pathways, respectively.
Potter
L R
LR
Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee 37232.
Garbers
D L
DL
D016475:3T3 Cells; D000818:Animals; D009320:Atrial Natriuretic Factor / Q000494:pharmacology*; D002462:Cell Membrane / Q000378:metabolism; D006152:Cyclic GMP / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D003995:Dibutyryl Cyclic GMP / Q000494:pharmacology; D004789:Enzyme Activation; D006162:Guanylate Cyclase / Q000378:metabolism*; D015151:Immunoblotting; D051379:Mice; D010449:Peptide Mapping; D010748:Phosphopeptides / Q000737:chemistry / Q000302:isolation & purification; D011493:Protein Kinase C / Q000378:metabolism*; D011869:Radioligand Assay; D051381:Rats; D017461:Receptors, Atrial Natriuretic Factor / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D013873:Thionucleotides / Q000494:pharmacology; D014162:Transfection
D010748:Phosphopeptides; D013873:Thionucleotides; C016276:8-bromocyclic GMP; D003995:Dibutyryl Cyclic GMP; C055656:8-((4-chlorophenyl)thio)cyclic-3',5'-GMP; D009320:Atrial Natriuretic Factor; D011493:Protein Kinase C; D006162:Guanylate Cyclase; D017461:Receptors, Atrial Natriuretic Factor; D006152:Cyclic GMP; D013755:Tetradecanoylphorbol Acetate