Related Experiment Videos
Differential phosphorylation paradigms dictate desensitization and internalization of the N-formyl peptide receptor
D C Maestes1, R M Potter, E R Prossnitz
1Department of Cell Biology, University of New Mexico Health Science Center, Albuquerque, New Mexico 87131, USA.
Abstract:
Following activation by ligand, most G protein-coupled receptors undergo rapid phosphorylation. This is accompanied by a drastic decrease in the efficacy of continued or repeated stimulation, due to receptor uncoupling from G protein and receptor internalization. Such processing steps have been shown to be absolutely dependent on receptor phosphorylation in the case of the N-formyl peptide receptor (FPR). In this study, we report results that indicate that the mechanisms responsible for desensitization and internalization are distinct. Using site-directed mutagenesis of the serine and threonine residues of the FPR carboxyl terminus, we have characterized regions that differentially regulate these two processes. Whereas substitution of all 11 Ser/Thr residues in the carboxyl terminus prevents both desensitization and internalization, substitution of four Ser/Thr residues between 328-332 blocks desensitization but has no effect on internalization. Similarly, substitution of four Ser/Thr residues between positions 334 and 339 results in a deficit in desensitization but again no decrease in internalization, suggesting that phosphorylation at either site evokes receptor internalization, whereas maximal desensitization requires phosphorylation at both sites. These results also indicate that receptor internalization is not involved in the process of desensitization. Further analysis of the residues between 328-332 revealed that restoration either of Ser(328) and Thr(329) or of Thr(331) and Ser(332) was sufficient to restore desensitization, suggesting that phosphorylation within either of these two sites, in addition to sites between residues 334 and 339, is sufficient to produce desensitization. Taken together, these results indicate that the mechanisms involved in FPR processing (uncoupling from G proteins and internalization) are regulated differentially by phosphorylation at distinct sites within the carboxyl terminus of the FPR. The relevance of this paradigm to other G protein-coupled receptors is discussed.
Insights
G protein-coupled receptor (GPCR) phosphorylation distinct sites regulate desensitization and internalization. Specific serine/threonine residues on the N-formyl peptide receptor (FPR) carboxyl terminus differentially control these processes.
Area of Science:
- Molecular Pharmacology
- Cell Biology
- Signal Transduction
Background:
- G protein-coupled receptors (GPCRs) are crucial cell surface receptors involved in numerous physiological processes.
- Upon ligand binding, GPCRs undergo phosphorylation, leading to desensitization and internalization, which limits cellular response.
- The N-formyl peptide receptor (FPR) is a key GPCR involved in inflammatory responses, and its processing is phosphorylation-dependent.
Purpose of the Study:
- To investigate the distinct mechanisms regulating desensitization and internalization of the N-formyl peptide receptor (FPR).
- To identify specific phosphorylation sites within the FPR carboxyl terminus that differentially control these processes.
Main Methods:
- Site-directed mutagenesis of serine and threonine residues in the FPR carboxyl terminus.
- Analysis of receptor desensitization and internalization following mutations.
Main Results:
- Mutating all 11 Ser/Thr residues abolished both desensitization and internalization.
- Mutations in residues 328-332 or 334-339 impaired desensitization but not internalization.
- Phosphorylation at either site (328-332 or 334-339) was sufficient for internalization, while maximal desensitization required phosphorylation at both sites.
- Receptor internalization was found to be independent of desensitization.
Conclusions:
- FPR desensitization and internalization are distinct processes regulated by phosphorylation at different sites within the carboxyl terminus.
- Specific phosphorylation sites differentially control FPR uncoupling from G proteins and receptor internalization.
- This phosphorylation-dependent differential regulation paradigm may apply to other GPCRs.