Agonist-induced endocytosis of lysophosphatidic acid-coupled LPA1/EDG-2 receptors via a dynamin2- and Rab5-dependent pathway

J Cell Sci. 2003 May 15;116(Pt 10):1969-80. doi: 10.1242/jcs.00397. Epub 2003 Mar 26.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • DNA / metabolism
  • Dose-Response Relationship, Drug
  • Dynamin II / metabolism*
  • Endocytosis*
  • Epitopes
  • Fluorescent Antibody Technique, Indirect
  • HeLa Cells
  • Humans
  • Immunoblotting
  • Lysophospholipids / metabolism*
  • Mitogen-Activated Protein Kinase 1 / metabolism
  • Mitogen-Activated Protein Kinase 3
  • Mitogen-Activated Protein Kinases / metabolism
  • Protein Structure, Tertiary
  • Receptors, G-Protein-Coupled / agonists
  • Receptors, G-Protein-Coupled / metabolism*
  • Receptors, Lysophosphatidic Acid
  • Serum Response Factor / metabolism
  • Time Factors
  • Transfection
  • rab5 GTP-Binding Proteins / metabolism*

Substances

  • Epitopes
  • Lysophospholipids
  • Receptors, G-Protein-Coupled
  • Receptors, Lysophosphatidic Acid
  • Serum Response Factor
  • DNA
  • Mitogen-Activated Protein Kinase 1
  • Mitogen-Activated Protein Kinase 3
  • Mitogen-Activated Protein Kinases
  • rab5 GTP-Binding Proteins
  • Dynamin II