Role of the Rab11-associated intracellular pool of receptors formed by constitutive endocytosis of the beta isoform of the thromboxane A2 receptor (TP beta).
Intracellular trafficking pathways of G protein-coupled receptors (GPCRs), following their agonist-induced endocytosis and their consequences on receptor function, are the subject of intense research efforts. However, less is known regarding their constitutive endocytosis. We previously demonstrated that the beta isoform of the thromboxane A(2) receptor (TPbeta) undergoes constitutive and agonist-induced endocytosis. Constitutive endocytosis of GPCRs can lead to the formation of an intracellular pool of receptors from which they can recycle back to the cell surface. In the present report, we show with the help of two TPbeta mutants (TPbeta-Y339A and TPbeta-I343A) specifically deficient in constitutive endocytosis that this intracellular pool of receptors serves to maintain agonist sensitivity over prolonged receptor stimulation in HEK293 cells. Second messenger generation by the TPbeta-Y339A and TPbeta-I343A mutants was drastically reduced compared to the wild-type receptor as suggested by dose-response and time-course experiments of inositol phosphates production following agonist treatment, despite normal coupling between the receptors and the Galpha(q) protein. Moreover, second messenger production after receptor activation was dramatically reduced when cells were pretreated with monensin, a recycling inhibitor. Receptor cell surface expression and endocytosis experiments further revealed that the small GTPase Rab11 protein is a determinant factor in controlling TPbeta recycling back to the cell surface. Co-localization experiments performed by immunofluorescence microscopy indicated that both constitutive and agonist-triggered endocytosis resulted in targeting of TPbeta to the Rab11-positive recycling endosome. Thus, we provide evidence that constitutive endocytosis of TPbeta forms a pool of receptors in the perinuclear recycling endosome from which they recycle to the cell surface, a process involved in preserving receptor sensitivity to agonist stimulation.
Thériault
Caroline
C
Service de Rhumatologie, Faculté de Médecine and Centre de Recherche Clinique-CHUS, Université de Sherbrooke, Sherbrooke, Québec, Canada, J1H 5N4.
Rochdi
Moulay Driss
MD
Parent
Jean-Luc
JL
D000409:Alanine / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics; D002460:Cell Line; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism; D004705:Endocytosis / Q000235:genetics; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007295:Inositol Phosphates / Q000096:biosynthesis; D007424:Intracellular Fluid / Q000378:metabolism* / Q000502:physiology; D007532:Isoleucine / Q000235:genetics; D020033:Protein Isoforms / Q000819:agonists / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport / Q000235:genetics; D044244:Receptors, Thromboxane A2, Prostaglandin H2 / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D015290:Second Messenger Systems / Q000235:genetics; D014162:Transfection; D014443:Tyrosine / Q000235:genetics; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D000099193:rab11 GTP-Binding Proteins
D000409:Alanine; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D007295:Inositol Phosphates; D007532:Isoleucine; D020033:Protein Isoforms; D044244:Receptors, Thromboxane A2, Prostaglandin H2; D014443:Tyrosine; D020691:rab GTP-Binding Proteins; D000099193:rab11 GTP-Binding Proteins