Internalization of the human N-formyl peptide and C5a chemoattractant receptors occurs via clathrin-independent mechanisms.
After stimulation by ligand, most G protein-coupled receptors (GPCRs) undergo rapid phosphorylation, followed by desensitization and internalization. In the case of the N-formyl peptide receptor (FPR), these latter two processing steps have been shown to be entirely dependent on phosphorylation of the receptor's carboxy terminus. We have previously demonstrated that FPR internalization can occur in the absence of receptor desensitization, indicating that FPR desensitization and internalization are regulated differentially. In this study, we have investigated whether human chemoattractant receptors internalize via clathrin-coated pits. Internalization of the FPR transiently expressed in HEK 293 cells was shown to be dependent upon receptor phosphorylation. Despite this, internalization of the FPR, as well as the C5a receptor, was demonstrated to be independent of the actions of arrestin, dynamin, and clathrin. In addition, we utilized fluorescence microscopy to visualize the FPR and beta(2)-adrenergic receptor as they internalized in the same cell, revealing distinct sites of internalization. Last, we found that a nonphosphorylatable mutant of the FPR, unable to internalize, was competent to activate p44/42 MAP kinase. Together, these results demonstrate not only that the FPR internalizes via an arrestin-, dynamin-, and clathrin-independent pathway but also that signal transduction to MAP kinases occurs in an internalization-independent manner.
Gilbert
T L
TL
Department of Cell Biology and Physiology, University of New Mexico Health Science Center, Albuquerque, New Mexico 87131, USA.
Bennett
T A
TA
Maestas
D C
DC
Cimino
D F
DF
Prossnitz
E R
ER
D015703:Antigens, CD / Q000378:metabolism*; D019393:Arrestin / Q000378:metabolism; D002460:Cell Line; D002966:Clathrin / Q000235:genetics / Q000502:physiology*; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism; D015936:Complement C5a / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D034281:Dynamins; D004789:Enzyme Activation; D005434:Flow Cytometry; D020558:GTP Phosphohydrolases / Q000378:metabolism; D018922:HL-60 Cells; D006801:Humans; D008024:Ligands; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D044087:Receptor, Anaphylatoxin C5a; D018343:Receptors, Adrenergic, beta-2 / Q000378:metabolism; D011951:Receptors, Complement / Q000378:metabolism*; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D018000:Receptors, Peptide / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D051552:beta-Adrenergic Receptor Kinases
D015703:Antigens, CD; D019393:Arrestin; D002966:Clathrin; D008024:Ligands; D044087:Receptor, Anaphylatoxin C5a; D018343:Receptors, Adrenergic, beta-2; D011951:Receptors, Complement; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic; D018000:Receptors, Peptide; D009240:N-Formylmethionine Leucyl-Phenylalanine; D015936:Complement C5a; D017868:Cyclic AMP-Dependent Protein Kinases; D051552:beta-Adrenergic Receptor Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D020558:GTP Phosphohydrolases; D034281:Dynamins