Agonist-induced endocytosis of lysophosphatidic acid-coupled LPA1/EDG-2 receptors via a dynamin2- and Rab5-dependent pathway.
Lysophosphatidic acid (LPA) is a serum-borne phospholipid that exerts a pleiotropic range of effects on cells through activation of three closely related G-protein-coupled receptors termed LPA1/EDG-2, LPA2/EDG-4 and LPA3/EDG-7. Of these receptors, the LPA1 receptor is the most widely expressed. In this study, we investigated the agonist-induced endocytosis of the human LPA1 receptor, bearing an N-terminal FLAG epitope tag, in stably transfected HeLa cells. Treatment with LPA induced the rapid endocytosis of approximately 40% of surface LPA1 within 15 minutes. Internalization was both dose dependent and LPA specific since neither lysophophatidylcholine nor sphingosine-1-phosphate induced LPA1 endocytosis. Removal of agonist following 30 minutes incubation resulted in recycling of LPA1 back to the cell surface. LPA1 internalization was strongly inhibited by dominant-inhibitory mutants of both dynamin2 (K44A) and Rab5a (S34N). In addition, both dynamin2 K44A and Rab5 S34N mildly inhibited LPA1-dependent activation of serum response factor. Finally, our results also indicate that LPA1 exhibits basal, LPA-dependent internalization in the presence of serum-containing medium.
Murph
Mandi M
MM
School of Biology and Petit Institute for Biosciences and Bioengineering, Georgia Institute of Technology, Atlanta, GA 30332-0363, USA.
Scaccia
Launa A
LA
Volpicelli
Laura A
LA
Radhakrishna
Harish
H
D004247:DNA / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D034285:Dynamin II / Q000378:metabolism*; D004705:Endocytosis; D000939:Epitopes; D019084:Fluorescent Antibody Technique, Indirect; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D008246:Lysophospholipids / Q000378:metabolism*; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D017434:Protein Structure, Tertiary; D043562:Receptors, G-Protein-Coupled / Q000819:agonists / Q000378:metabolism*; D049368:Receptors, Lysophosphatidic Acid; D026362:Serum Response Factor / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D020696:rab5 GTP-Binding Proteins / Q000378:metabolism*
D000939:Epitopes; D008246:Lysophospholipids; D043562:Receptors, G-Protein-Coupled; D049368:Receptors, Lysophosphatidic Acid; D026362:Serum Response Factor; D004247:DNA; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D020696:rab5 GTP-Binding Proteins; D034285:Dynamin II