The regions within the N-terminus critical for human glucagon like peptide-1 receptor (hGLP-1R) cell Surface expression

Abstract

The hGLP-1R is a target for the treatment of type 2 diabetes and belongs to the class B family of GPCRs. Like other class B GPCRs, the GLP-1R contains an N-terminal signal peptide (SP) and undergoes N-linked glycosylation, which are important for its trafficking and maturation. This study analysed the role of the SP, the hydrophobic region after the SP (HRASP), glycosylation and the conserved residues within the N-terminus in GLP-1R trafficking. HGLP-1R targeted to the cell surface showed no SP, and the SP deleted mutant, but not the mutants defective in SP cleavage, showed cell surface expression, demonstrating the importance of SP cleavage for hGLP-1R cell surface expression. The N-terminal deletions of hGLP-1R revealed that the HRASP, not the SP, is essential for cell surface expression of GLP-1R. Further, inhibition of hGLP-1R glycosylation prevented cell surface expression of the receptor. Mutation of Trp39, Tyr69 and Tyr88, which are required for agonist binding, in the GLP-1R abolished cell surface expression of the receptor independent of the SP cleavage or N-linked glycosylation. In conclusion, the N-terminus of hGLP-1R regulates receptor trafficking and maturation. Therefore this study provides insight into the role of hGLP-1R N-terminus on the receptor cell surface expression.

Luteinizing hormone/chorionic gonadotrophin receptor overexpressed in granulosa cells from polycystic ovary syndrome ovaries is functionally active

“…The protein content of cell lysates was estimated using the bicinchoninic acid (BCA) assay (Sigma-Aldrich, Dorset, UK) and BSA as a protein standard (Thompson and Kanamarlapudi, 2014).…”

Distinct regions in the C-Terminus required for GLP-1R cell surface expression, activity and internalisation

“…The high molecular weight band in the doublet has previously been shown as the mature form of the receptor whereas the low molecular weight band represents the immature form of the receptor (Thompson and Kanamarlapudi, 2014;Widmann et al, 1995). However, the N410 construct only showed a single band, which corresponds to the lower molecular weight band of the doublet, with both the antibodies, demonstrating its existing mainly as the immature form of the receptor (Fig.…”

“…However, three deletion mutations, D411e418, D419e430 and D431e450, and a site-directed mutant (E408A, V409A, Q410A), which has previously been shown to affect agonist binding to the hGLP-1R (Vazquez et al, 2005a), contain an additional GFP-tag at the C-terminus. We have shown previously that the cAMP producing activity of the SP-VSVG-GFP construct (which contains both VSVG and GFP tags) is similar to that of the hGLP-1R with no tag or either of the VSVG-tag or GFP-tag, indicating that the attachment of the VSVG and GFP tags to the hGLP-1R had no effect on the activity of the receptor (Thompson and Kanamarlapudi, 2014).…”

“…Like other family B GPCRs, the GLP-1R contains an N-terminal signal peptide (SP) and undergoes N-linked glycosylation, which are important for the newly synthesised receptor cell surface expression (Huang et al, 2010;Chen et al, 2010;Whitaker et al, 2012). We have recently demonstrated the importance of SP cleavage, N-linked glycosylation and the hydrophobic region after the SP (HRASP) within the Nterminus of the GLP-1R for its biosynthetic trafficking to the plasma membrane (Thompson and Kanamarlapudi, 2014). The intracellular C-terminal domain of GPCRs plays a critical role in agonist-induced internalisation, desensitisation, down regulation and arrestin signalling of the receptors (Thompson et al, 2008).…”

Glucagon‐like peptide‐1 receptor undergoes importin‐α‐dependent nuclear localization in rat aortic smooth muscle cells

“…Like other proteins in the family B of G protein‐coupled receptors, GLP‐1R contains a large hydrophilic extracellular domain, an intracellular C‐terminal domain and seven transmembrane helices. GLP‐1R is expressed on the cell surface and the N‐terminal sequence after signal peptide (SP) plays a key role in receptor activation . Distinct regions within the C‐terminal domain of GLP‐1R are critical for cell surface expression (411‐418aa), cAMP production (419‐430aa) and agonist‐induced internalization (431‐450aa) of the receptor .…”