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 LPA 1 /EDG-2, LPA 2 /EDG-4 and LPA 3 /EDG-7. Of these receptors, the LPA 1 receptor is the most widely expressed. In this study, we investigated the agonist-induced endocytosis of the human LPA 1 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 LPA 1 within 15 minutes. Internalization was both dose dependent and LPA specific since neither lysophophatidylcholine nor sphingosine-1-phosphate induced LPA 1 endocytosis. Removal of agonist following 30 minutes incubation resulted in recycling of LPA 1 back to the cell surface. LPA 1 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 LPA 1 -dependent activation of serum response factor. Finally, our results also indicate that LPA 1 exhibits basal, LPA-dependent internalization in the presence of serum-containing medium.