The glucagon-like peptide-2 receptor C terminus modulates beta-arrestin-2 association but is dispensable for ligand-induced desensitization, endocytosis, and G-protein-dependent effector activation.
Classic models of receptor desensitization and internalization have been largely based on the behavior of Family A G-protein-coupled receptors (GPCRs). The glucagon-like peptide-2 receptor (GLP-2R) is a member of the Family B glucagon-secretin GPCR family, which exhibit significant sequence and structural differences from the Family A receptors in their intracellular and extracellular domains. To identify structural motifs that regulate GLP-2R signaling and cell surface receptor expression, we analyzed the functional properties of a series of mutant GLP-2Rs. The majority of the C-terminal receptor tail was dispensable for GLP-2-induced cAMP accumulation, ERK1/2 activation, and endocytosis in transfected cells. However, progressive truncation of the C terminus reduced cell surface receptor expression, altered agonist-induced GLP-2R trafficking, and abrogated protein kinase A-mediated heterologous receptor desensitization. Elimination of the distal 21 amino acids of the receptor was sufficient to promote constitutive receptor internalization and prevent agonist-induced recruitment of beta-arrestin-2. Site-directed mutagenesis identified specific amino acid residues within the distal GLP-2R C terminus that mediate the stable association with beta-arrestin-2. Surprisingly, although the truncated mutant receptors failed to interact with beta-arrestin-2, they underwent homologous desensitization and subsequent resensitization with kinetics similar to that observed with the wild-type GLP-2R. Our data suggest that, although the GLP-2R C terminus is not required for coupling to cellular machinery regulating signaling or desensitization, it may serve as a sorting signal for intracellular trafficking. Taken together with the previously demonstrated clathrin and dynamin-independent, lipid-raft-dependent pathways for internalization, our data suggest that GLP-2 receptor signaling has evolved unique structural and functional mechanisms for control of receptor trafficking, desensitization, and resensitization.
Estall
Jennifer L
JL
Departments of Laboratory Medicine and Pathobiology, and Medicine, University of Toronto, The Banting and Best Diabetes Centre, Toronto General Hospital, University of Toronto, Toronto M5G 2C4, Canada.
Koehler
Jacqueline A
JA
Yusta
Bernardo
B
Drucker
Daniel J
DJ
D000262:Adenylyl Cyclases / Q000378:metabolism; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D019390:Arrestins / Q000378:metabolism*; D018929:Cell Culture Techniques; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D002966:Clathrin / Q000737:chemistry; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004705:Endocytosis; D019204:GTP-Binding Proteins / Q000378:metabolism; D000067757:Glucagon-Like Peptide-1 Receptor; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008024:Ligands; D021962:Membrane Microdomains / Q000737:chemistry; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011493:Protein Kinase C / Q000378:metabolism; D017434:Protein Structure, Tertiary; D018027:Receptors, Glucagon / Q000737:chemistry* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D013997:Time Factors; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins
C000605254:ARRB2 protein, human; D019390:Arrestins; D002966:Clathrin; D018076:DNA, Complementary; C000593833:GLP1R protein, human; D000067757:Glucagon-Like Peptide-1 Receptor; D008024:Ligands; D018027:Receptors, Glucagon; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins; D000242:Cyclic AMP; D011493:Protein Kinase C; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases