Regulation of N-formyl peptide receptor signaling and trafficking by individual carboxyl-terminal serine and threonine residues.
Adaptation, defined as the diminution of receptor signaling in the presence of continued or repeated stimulation, is critical to cellular function. G protein-coupled receptors (GPCRs) undergo multiple adaptive processes, including desensitization and internalization, through phosphorylation of cytoplasmic serine and threonine residues. However, the relative importance of individual and combined serine and threonine residues to these processes is not well understood. We examined this mechanism in the context of the N-formyl peptide receptor (FPR), a well-characterized member of the chemoattractant/chemokine family of GPCRs critical to neutrophil function. To evaluate the contributions of individual and combinatorial serine and threonine residues to internalization, desensitization, and arrestin2 binding, 30 mutant forms of the FPR, expressed in the human promyelocytic U937 cell line, were characterized. We found that residues Ser(328), Ser(332), and Ser(338) are individually critical, and indeed sufficient, for internalization, desensitization, and arrestin2 binding, but that the presence of neighboring threonine residues can inhibit these processes. Additionally, we observed no absolute correlation between arrestin binding and either internalization or desensitization, suggesting the existence of arrestin-independent mechanisms for these processes. Our results suggest C-terminal serine and threonine residues of the FPR represent a combinatorial code, capable of both positively and negatively regulating signaling and trafficking. This study is among the first detailed analyses of a complex regulatory site in a GPCR, and provides insight into GPCR regulatory mechanisms.
Potter
Ross M
RM
Department of Cell Biology and Physiology, University of New Mexico Cancer Research and Treatment Center, University of New Mexico Health Sciences Center, Albuquerque, NM 87131, USA.
Maestas
Diane C
DC
Cimino
Daniel F
DF
Prossnitz
Eric R
ER
D002460:Cell Line; D006801:Humans; D009154:Mutation / Q000235:genetics; D021381:Protein Transport; D044042:Receptors, Formyl Peptide / Q000235:genetics / Q000378:metabolism*; D012694:Serine / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D013912:Threonine / Q000235:genetics / Q000378:metabolism*
D044042:Receptors, Formyl Peptide; D013912:Threonine; D012694:Serine