Phosphorylation of the prostacyclin receptor during homologous desensitization. A critical role for protein kinase c.
Agonist-induced phosphorylation of an epitope-tagged prostacyclin receptor (HAhIP) is mediated primarily by PKC (Smyth, E. M., Nestor, P. V., and FitzGerald G. A. (1996) J. Biol. Chem. 271, 33698-33704). Based on the two consensus sites for protein kinase C (PKC) phosphorylation in the C-terminal region mutant HAhIPs were generated: S328A and S374A, in which an alanine replaced Ser-328 or Ser-374, respectively, S328A/S374A and C-DEL, in which the C-terminal portion was truncated at amino acid 313. Mutant receptors, stably expressed in HEK293 cells, coupled normally to cAMP production. Substantially less coupling to inositol phosphate was apparent with S328A, S328A/S374A, and C-DEL compared with HAhIP or S374A. Point mutants resolved by SDS-polyacrylamide gel electrophoresis as a broad band with a molecular mass of 44-62, indicating that the receptors are glycosylated, and immunofluoresence staining demonstrated their membrane localization. C-DEL demonstrated a substantial reduction in glycosylation; bands with molecular masses of 38-54 (glycosylated), 30, and 27 kDa (unglycosylated) were apparent. Although membrane localization was evident, cellular localization was more diffuse. HAhIP and S374A underwent iloprost- and PMA-induced phosphorylation (1 and 5 microM, respectively, for 10 min). S328A and S328A/S374A showed a markedly less iloprost- and no PMA-induced phosphorylation. Phosphorylation of C-DEL was completely absent with either agonist. Electrospray mass spectrometry indicated that a peptide, including Ser-328, was phosphorylated in vitro by PKC, whereas one including Ser-374 was not. Iloprost (1 microM, 10 min) desensitized HAhIP- and S374A-mediated adenylyl cyclase activation. A less impressive desensitization was evident with S328A and S328A/S374A, and no desensitization of C-DEL coupling was apparent. Exposure of transfected cells to iloprost (1 microM) for increasing times induced a rapid desensitization of subsequent iloprost-induced (1 microM) HAhIP and S374A adenylyl cyclase coupling. In contrast, no significant time-dependent desensitization of S328A, S328A/S374A, or C-DEL coupling was evident. These results indicate that PKC-dependent phosphorylation is of critical importance to homologous regulation of hIP. Ser-328 is a primary site for PKC phosphorylation of hIP.
Center for Experimental Therapeutics, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
E M
EM
Smyth
W H
WH
Li
G A
GA
FitzGerald
D000262:Adenylyl Cyclases / Q000378:metabolism; D000595:Amino Acid Sequence; D002451:Cell Compartmentation; D000242:Cyclic AMP / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006801:Humans; D016285:Iloprost / Q000494:pharmacology; D007295:Inositol Phosphates / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D044006:Receptors, Epoprostenol; D011982:Receptors, Prostaglandin / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D015290:Second Messenger Systems; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D007295:Inositol Phosphates; D044006:Receptors, Epoprostenol; D011982:Receptors, Prostaglandin; D011994:Recombinant Proteins; D000242:Cyclic AMP; D011493:Protein Kinase C; D000262:Adenylyl Cyclases; D016285:Iloprost; D013755:Tetradecanoylphorbol Acetate