Involvement of actin in agonist-induced endocytosis of the G protein-coupled receptor for thromboxane A2: overcoming of actin disruption by arrestin-3 but not arrestin-2.
The role of actin in endocytosis of G protein-coupled receptors is poorly defined. In the present study, we demonstrate that agents that depolymerize (latrunculin B and cytochalasin D) or stabilize (jasplakinolide) the actin cytoskeleton blocked agonist-induced endocytosis of the beta isoform of the thromboxane A(2) receptor (TPbeta) in HEK293 cells. This suggests that endocytosis of TPbeta requires active remodeling of the actin cytoskeleton. On the other hand, disruption of microtubules with colchicine did not affect endocytosis of the receptor. Expression of wild-type and mutant forms of the small GTPases RhoA and Cdc42 potently inhibited endocytosis of TPbeta, further indicating a role for the dynamic regulation of the actin cytoskeleton in this pathway. Agonist treatment of TPbeta in HEK293 cells resulted in the formation of actin stress fibers through Galpha(q/11) signaling. Because we previously showed that endocytosis of TPbeta is dependent on arrestins, we decided to explore the relation between arrestin-2 and -3 and actin in endocytosis of this receptor. Interestingly, we show that the inhibition of TPbeta endocytosis by the actin toxins in HEK293 cells was overcome by the overexpression of arrestin-3, but not of arrestin-2. These results indicate that the actin cytoskeleton is not essential in arrestin-3-mediated endocytosis of TPbeta. However, arrestin-3 could not promote endocytosis of the TPbetaY339A and TPbetaI343A carboxyl-terminal mutants when the actin cytoskeleton was disrupted. Our data provide new evidence that the actin cytoskeleton plays an essential role in TPbeta endocytosis. Furthermore, our work suggests the existence of actin-dependent and -independent arrestin-mediated pathways of endocytosis.
Laroche
Geneviève
G
Service de Rhumatologie, Département de Médecine, Faculté de Médecine and Centre de Recherche Clinique, Université de Sherbrooke, Quebec, Canada.
Rochdi
Moulay Driss
MD
Laporte
Stéphane A
SA
Parent
Jean-Luc
JL
D000199:Actins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D000970:Antineoplastic Agents / Q000494:pharmacology; D019390:Arrestins / Q000502:physiology*; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D002460:Cell Line; D002966:Clathrin / Q000378:metabolism; D003001:Cloning, Molecular; D003078:Colchicine / Q000494:pharmacology; D015638:Cytochalasin D / Q000494:pharmacology; D003599:Cytoskeleton / Q000378:metabolism; D047630:Depsipeptides / Q000494:pharmacology; D004705:Endocytosis; D004797:Enzyme-Linked Immunosorbent Assay; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000378:metabolism; D006801:Humans; D008387:Marine Toxins / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D010750:Phosphoproteins / Q000502:physiology*; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D044244:Receptors, Thromboxane A2, Prostaglandin H2 / Q000737:chemistry* / Q000378:metabolism*; D015398:Signal Transduction; D013844:Thiazoles / Q000494:pharmacology; D053778:Thiazolidines; D013997:Time Factors; D014162:Transfection; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000737:chemistry / Q000378:metabolism
D000199:Actins; D000970:Antineoplastic Agents; D019390:Arrestins; D019086:Bridged Bicyclo Compounds, Heterocyclic; D002966:Clathrin; D047630:Depsipeptides; D008387:Marine Toxins; D019384:Nucleic Acid Synthesis Inhibitors; D010750:Phosphoproteins; D043562:Receptors, G-Protein-Coupled; D044244:Receptors, Thromboxane A2, Prostaglandin H2; D013844:Thiazoles; D053778:Thiazolidines; C082333:arrestin3; C057531:jasplakinolide; D015638:Cytochalasin D; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D020764:cdc42 GTP-Binding Protein; D020742:rhoA GTP-Binding Protein; C037068:latrunculin B; D003078:Colchicine