Phosphorylation of the prostacyclin receptor during homologous desensitization - A critical role for protein kinase C

被引:58
作者
Smyth, EM [1 ]
Li, WH [1 ]
Fitzgerald, GA [1 ]
机构
[1] Univ Penn, Ctr Expt Therapeut, Stellar Chance Labs 905, Philadelphia, PA 19104 USA
关键词
D O I
10.1074/jbc.273.36.23258
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Agonist-induced phosphorylation of an epitope-tagged prostacyclin receptor (HAhIP) is mediated primarily by PRC (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 mu M, 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 mu M, 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 mu M) for increasing times induced a rapid desensitization of subsequent iloprost-induced (1 mu M) 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.
引用
收藏
页码:23258 / 23266
页数:9
相关论文
共 28 条
  • [1] The long cytoplasmic carboxyl terminus of the prostaglandin E(2) receptor EP(4) subtype is essential for agonist-induced desensitization
    Bastepe, M
    Ashby, B
    [J]. MOLECULAR PHARMACOLOGY, 1997, 51 (02) : 343 - 349
  • [2] BOIE Y, 1994, J BIOL CHEM, V269, P12173
  • [3] Switching of the coupling of the beta(2)-adrenergic receptor to different G proteins by protein kinase A
    Daaka, Y
    Luttrell, LM
    Lefkowitz, RJ
    [J]. NATURE, 1997, 390 (6655) : 88 - 91
  • [4] FISCH A, 1997, CIRCULATION, V96, P765
  • [5] Freedman NJ, 1996, RECENT PROG HORM RES, V51, P319
  • [6] PHOSPHORYLATION AND DESENSITIZATION OF THE HUMAN BETA(1)-ADRENERGIC RECEPTOR - INVOLVEMENT OF G-PROTEIN-COUPLED RECEPTOR KINASES AND CAMP-DEPENDENT PROTEIN-KINASE
    FREEDMAN, NJ
    LIGGETT, SB
    DRACHMAN, DE
    PEI, G
    CARON, MG
    LEFKOWITZ, RJ
    [J]. JOURNAL OF BIOLOGICAL CHEMISTRY, 1995, 270 (30) : 17953 - 17961
  • [7] Internalization and down-regulation of the prostacyclin receptor in human platelets
    Giovanazzi, S
    Accomazzo, MR
    Letari, O
    Oliva, D
    Nicosia, S
    [J]. BIOCHEMICAL JOURNAL, 1997, 325 : 71 - 77
  • [8] G-protein-coupled receptor kinase activity is increased in hypertension
    Gros, R
    Benovic, JL
    Tan, CM
    Feldman, RD
    [J]. JOURNAL OF CLINICAL INVESTIGATION, 1997, 99 (09) : 2087 - 2093
  • [9] Rapid, agonist-dependent phosphorylation in vivo of human thromboxane receptor isoforms - Minimal involvement of protein kinase C
    Habib, A
    Vezza, R
    Creminon, C
    Maclouf, J
    FitzGerald, GA
    [J]. JOURNAL OF BIOLOGICAL CHEMISTRY, 1997, 272 (11) : 7191 - 7200
  • [10] HAUSDORFF WP, 1990, SYM SOC EXP BIOL, V44, P225