The cytoplasmic domain close to the transmembrane region of the glucagon-like peptide-1 receptor contains sequence elements that regulate agonist-dependent internalisation.
In order to gain better insight into the molecular events involved in the signal transduction generated through glucagon-like peptide-1 (GLP-1) receptors, we tested the effect of deletions and point mutations within the cytoplasmic tail of this receptor with a view to establishing relationships between signal transduction desensitisation and receptor internalisation. Wild-type and truncated (deletion of the last 27 amino acids (GLPR 435R) and deletion of 44 amino acids (GLPR 418R)) GLP-1 receptors bound the agonist with similar affinity. Deletion of the last 27 amino acids decreased the internalisation rate by 78%, while deletion of 44 amino acids containing all the phosphorylation sites hitherto described in this receptor decreased the internalisation rate by only 47%. Binding of the ligand to both receptors stimulated adenylyl cyclase. In contrast, deletion of the region containing amino acids 419 to 435 (GLPR 419delta435) increased the internalisation rate by 268%, and the replacement of EVQ(408-410) by alanine (GLPR A(408-410)) increased this process to 296%. In both receptors, the efficacy in stimulating adenylate cyclase was decreased. All the receptors studied were internalised by coated pits, except for the receptor with a deletion of the last 44 amino acids, which also had a faster resensitisation rate. Our findings indicate that the neighbouring trans-membrane domain of the carboxyl-terminal tail of the GLP-1 receptor contains sequence elements that regulate agonist-dependent internalisation and transmembrane signalling.
Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Universidad Complutense, Ciudad Universitaria, 28040 Madrid, Spain.
Patricia
P
Vázquez
Isabel
I
Roncero
Enrique
E
Blázquez
Elvira
E
Alvarez
D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004705:Endocytosis; D017353:Gene Deletion; D005934:Glucagon / Q000378:metabolism; D052216:Glucagon-Like Peptide 1; D000067757:Glucagon-Like Peptide-1 Receptor; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D017354:Point Mutation; D011485:Protein Binding; D011498:Protein Precursors / Q000378:metabolism; D051381:Rats; D018027:Receptors, Glucagon / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction
C000593835:Glp1r protein, rat; D000067757:Glucagon-Like Peptide-1 Receptor; D010446:Peptide Fragments; D011498:Protein Precursors; D018027:Receptors, Glucagon; D052216:Glucagon-Like Peptide 1; D005934:Glucagon