The cytoplasmic domain close to the transmembrane region of the glucagon-like peptide-1 receptor contains sequence elements that regulate agonist-dependent internalisation

Abstract

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 419 435) increased the internalisation rate by 268%, and the replacement of EVQ [408][409][410] by alanine (GLPR A [408][409][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 transmembrane domain of the carboxyl-terminal tail of the GLP-1 receptor contains sequence elements that regulate agonist-dependent internalisation and transmembrane signalling.

Mechanisms of action of glucagon-like peptide 1 in the pancreas

“…Widmann and colleagues did not observe internalization of a mutant receptor lacking the last 33 amino acids (Widmann et al, 1997) while Vazquez (Vazquez et al, 2005a) showed a 78% slower internalization of a modified receptor lacking the last 27 amino acids when transfected into fibroblast cell lines. In contrast when the 44 C-terminal amino acids were deleted (GLPR 418R), receptor internalization was only 47% slower with the mutant versus the wild-type GLP-1R, indicating an inhibitory role of the region containing amino acids 419-435 (Vazquez et al, 2005a). Specifically, when the three amino acids located proximal to TM 7 ( 408 EVQ 410 ) were replaced with alanine, internalization was found to be much faster.…”

“…They have shown that GLP-1R is endocytosed via a primarily clathrin coated pitdependent mechanism and that in the presence of agonist the receptor cycles between the plasma membrane and endosomal compartments. The recognition sequence for the clathrin coated pit is located in the cytoplasmic tail of the receptor and C-terminally truncated mutants exhibit aberrant internalization rates (Vazquez et al, 2005a;Widmann et al, 1997). Widmann and colleagues did not observe internalization of a mutant receptor lacking the last 33 amino acids (Widmann et al, 1997) while Vazquez (Vazquez et al, 2005a) showed a 78% slower internalization of a modified receptor lacking the last 27 amino acids when transfected into fibroblast cell lines.…”

“…The recognition sequence for the clathrin coated pit is located in the cytoplasmic tail of the receptor and C-terminally truncated mutants exhibit aberrant internalization rates (Vazquez et al, 2005a;Widmann et al, 1997). Widmann and colleagues did not observe internalization of a mutant receptor lacking the last 33 amino acids (Widmann et al, 1997) while Vazquez (Vazquez et al, 2005a) showed a 78% slower internalization of a modified receptor lacking the last 27 amino acids when transfected into fibroblast cell lines. In contrast when the 44 C-terminal amino acids were deleted (GLPR 418R), receptor internalization was only 47% slower with the mutant versus the wild-type GLP-1R, indicating an inhibitory role of the region containing amino acids 419-435 (Vazquez et al, 2005a).…”

“…Specifically, when the three amino acids located proximal to TM 7 ( 408 EVQ 410 ) were replaced with alanine, internalization was found to be much faster. As approximately 40% of the GLPR 418R truncation was internalized when the cells were incubated in hypertonic media (which will disrupt clathrin coated pit-mediated endocytosis) it was postulated that this mutant receptor could be internalized via a faster, uncoated pit pathway (Vazquez et al, 2005a). A recent paper has shown evidence that GLP-1R may also undergo a caveolin-1-dependent trafficking to and from the cell membrane (Syme et al, 2006).…”

Genetic variation inGlp1rexpression influences the rate of gastric emptying in mice

“…The complete coding region for C57BL/6J and CAST/Ei Glp1r was subcloned into an expression plasmid pcDNA 3.1 (Invitrogen) and transfected into CHO-K1 cells (ATCC CCL-61; Ref. 56), graciously provided by Dr. Tom Gettys, using lipofectin (Invitrogen). Stable transfectants were obtained after selection for zeocin (zeo) resistance at a concentration of 0.5 mg/ml (Invitrogen) in DMEM containing 10% fetal bovine serum.…”