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Role of receptor phosphorylation in desensitization and internalization of the secretin receptor
M H Holtmann1, B F Roettger, D I Pinon
1Center for Basic Research in Digestive Diseases, Mayo Clinic and Foundation, Rochester, Minnesota 55905, USA.
Abstract:
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.
Insights
Secretin receptor desensitization involves two main mechanisms: rapid internalization and slower phosphorylation-dependent interference with G protein coupling, protecting cells from overstimulation.
Area of Science:
- Cellular signaling
- Molecular pharmacology
- G protein-coupled receptors
Background:
- The secretin receptor, a G protein-coupled receptor (GPCR), undergoes agonist-induced phosphorylation.
- C-terminal truncation prevents secretin receptor phosphorylation.
- Understanding desensitization mechanisms is crucial for GPCR signaling.
Purpose of the Study:
- To investigate the functional impact of secretin receptor phosphorylation on desensitization.
- To identify structural determinants of desensitization by comparing wild-type and truncated receptors.
- To elucidate the distinct roles of internalization and phosphorylation in receptor desensitization.
Main Methods:
- Utilized wild-type and C-terminally truncated secretin receptors expressed in cell lines.
- Developed a novel fluorescent secretin agonist for receptor internalization studies.
- Assessed cAMP accumulation and G protein coupling following agonist stimulation and desensitization protocols.
Main Results:
- Both wild-type and truncated receptors exhibited normal secretin binding, G protein coupling, and cAMP accumulation.
- Agonist exposure led to prompt receptor internalization for both constructs.
- Wild-type receptors showed absent cAMP responses post-desensitization, while truncated receptors had reduced responses, indicating phosphorylation-independent internalization as the primary mechanism.
- Phosphorylation was linked to a distinct process impairing G protein coupling, evidenced by a slower cAMP stimulation rate.
Conclusions:
- Secretin receptor desensitization is primarily mediated by phosphorylation-independent receptor internalization.
- Phosphorylation contributes to desensitization through a separate mechanism affecting G protein coupling efficiency.
- Dual desensitization pathways protect cells from excessive secretin receptor stimulation.