Role of receptor phosphorylation in desensitization and internalization of the secretin receptor.
The secretin receptor is prototypic of a recently described family of G protein-coupled receptors. We recently demonstrated its phosphorylation in response to agonist stimulation and elimination of this covalent modification by C-terminal truncation (F. Ozcelebi et al. (1995) Mol. Pharmacol. 48, 818-824). Here, we explore the functional impact of receptor phosphorylation and structural determinants for desensitization by comparing receptor behavior after agonist exposure in cell lines expressing wild-type and truncated receptor. To characterize receptor internalization, a novel fluorescent full agonist, [rat secretin-27]-Gly-rhodamine, was developed, which bound specifically and with high affinity. Both receptor constructs bound secretin normally, leading to normal G protein coupling and cAMP accumulation and prompt receptor internalization. Exposure to 10 nM secretin for 5 min or 12 h prior to washing and restimulation with a full range of concentrations demonstrated absent cAMP responses in wild-type receptor-bearing cells and responses 25 to 30% of control and shifted 1 order of magnitude to the right in the truncated receptor-bearing cells. Thus, the major mechanism of desensitization was phosphorylation-independent receptor internalization. Phosphorylation was associated with a distinct process that likely represents interference with G protein coupling, manifest as a reduced rate of cAMP stimulation. Thus, dual distinct mechanisms of desensitization exist in the secretin receptor family that should help protect receptor-bearing cells from overstimulation.
Holtmann
M H
MH
Center for Basic Research in Digestive Diseases, Mayo Clinic and Foundation, Rochester, Minnesota 55905, USA.
Roettger
B F
BF
Pinon
D I
DI
Miller
L J
LJ
D000818:Animals; D001692:Biological Transport; D016466:CHO Cells; D002451:Cell Compartmentation; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005456:Fluorescent Dyes; D019204:GTP-Binding Proteins / Q000378:metabolism; D007700:Kinetics; D010766:Phosphorylation; D043562:Receptors, G-Protein-Coupled; D011964:Receptors, Gastrointestinal Hormone / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012235:Rhodamines / Q000378:metabolism; D012633:Secretin / Q000031:analogs & derivatives / Q000378:metabolism*; D015398:Signal Transduction
C101783:(rat secretin-27)-Gly-rhodamine; D005456:Fluorescent Dyes; D043562:Receptors, G-Protein-Coupled; D011964:Receptors, Gastrointestinal Hormone; D011994:Recombinant Proteins; D012235:Rhodamines; C029937:secretin receptor; D012633:Secretin; D000242:Cyclic AMP; D019204:GTP-Binding Proteins