Lipid raft-dependent glucagon-like peptide-2 receptor trafficking occurs independently of agonist-induced desensitization.
The intestinotrophic and cytoprotective actions of glucagon-like peptide-2 (GLP-2) are mediated by the GLP-2 receptor (GLP-2R), a member of the class II glucagon-secretin G protein-coupled receptor superfamily. Although native GLP-2 exhibits a short circulating half-life, long-acting degradation-resistant GLP-2 analogues are being evaluated for therapeutic use in human subjects. Accordingly, we examined the mechanisms regulating signaling, internalization, and trafficking of the GLP-2R to identify determinants of receptor activation and desensitization. Heterologous cells expressing the transfected rat or human GLP-2R exhibited a rapid, dose-dependent, and prolonged desensitization of the GLP-2-stimulated cAMP response and a sustained GLP-2-induced decrease in levels of cell surface receptor. Surprisingly, inhibitors of clathrin-dependent endocytosis failed to significantly decrease GLP-2R internalization, whereas cholesterol sequestration inhibited ligand-induced receptor internalization and potentiated homologous desensitization. The hGLP-2R localized to both Triton X-100-soluble and -insoluble (lipid raft) cellular fractions and colocalized transiently with the lipid raft marker caveolin-1. Although GLP-2R endocytosis was dependent on lipid raft integrity, the receptor transiently associated with green fluorescent protein tagged-early endosome antigen 1-positive vesicles and inhibitors of endosomal acidification attenuated the reappearance of the GLP-2R on the cell surface. Our data demonstrate that GLP-2R desensitization and raft-dependent trafficking represent distinct and independent cellular mechanisms and provide new evidence implicating the importance of a clathrin- and dynamin-independent, lipid raft-dependent pathway for homologous G protein-coupled receptor internalization.
Estall
Jennifer L
JL
Department of Laboratory Medicine and Pathobiology, University of Toronto, The Banting and Best Diabetes Centre, Toronto General Hospital, Toronto, Canada M5G 2C4.
Yusta
Bernardo
B
Drucker
Daniel J
DJ
D000818:Animals; D051242:Caveolin 1; D022461:Caveolins / Q000032:analysis / Q000378:metabolism; D002784:Cholesterol / Q000378:metabolism; D002966:Clathrin / Q000378:metabolism; D006224:Cricetinae; D000242:Cyclic AMP / Q000096:biosynthesis; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology*; D011992:Endosomes / Q000187:drug effects / Q000276:immunology / Q000502:physiology; D005372:Filipin / Q000494:pharmacology; D053767:Glucagon-Like Peptide 2; D000067757:Glucagon-Like Peptide-1 Receptor; D004763:Glucagon-Like Peptides; D006801:Humans; D021962:Membrane Microdomains / Q000187:drug effects / Q000276:immunology / Q000378:metabolism*; D010455:Peptides / Q000494:pharmacology; D021381:Protein Transport / Q000502:physiology; D051381:Rats; D018027:Receptors, Glucagon / Q000819:agonists / Q000032:analysis / Q000378:metabolism*
C495540:CAV1 protein, human; C495537:Cav1 protein, rat; D051242:Caveolin 1; D022461:Caveolins; D002966:Clathrin; C000593833:GLP1R protein, human; C000593835:Glp1r protein, rat; D053767:Glucagon-Like Peptide 2; D000067757:Glucagon-Like Peptide-1 Receptor; D010455:Peptides; D018027:Receptors, Glucagon; D004763:Glucagon-Like Peptides; D005372:Filipin; D002784:Cholesterol; D000242:Cyclic AMP