The N-terminal Region of GLP-1R Regulates the Receptor Cell Surface Expression

Aiysha Thompson, Venkateswarlu Kanamarlapudi. Swansea University, Swansea, UK

Background:

Glucagon like Peptide-1 Receptor (GLP-1R) is a target for the treatment of type 2 diabetes and belongs to the class B family of G protein coupled receptors (GPCRs) (1). The class B GPCRs contain a putative N-terminal signal peptide (SP), which is important for trafficking of the receptor (2). Furthermore, GLP-1R undergoes N-linked glycosylation that plays an important role in receptor trafficking and maturation (3,4). The aim of this study was to analyse the role of the SP and glycosylation within the N-terminus in GLP-1R trafficking and agonist-induced internalisation. Additionally, the hydrophobic region after the SP (HRASP) was analysed for its effects on GLP-1R cell surface expression.

Methods: Cell surface expression, activity and agonist-induced internalisation of the N-terminal deleted and site-directed mutants of GLP-1R were examined using a number of biochemical techniques including ELISA, immunofluorescence, flow cytometry, western blotting and cAMP assays.

Results: GLP-1R targeted to the cell surface and showed no SP, indicating that it was cleaved off in the mature receptor (ELISA 0%; flow cytometry 1.1%; p<0.001). The SP deleted mutant (ΔSP) also showed cell surface expression (ELISA and flow cytometry 100%, p>0.05). However, two mutants defective in SP cleavage (VSP-ΔSP and A21R) showed no cell surface expression (ELISA 2.3% and 8.1%; flow cytometry 13.9% and 4.4%, p<0.001), demonstrating the importance of SP cleavage for GLP-1R cell surface expression. The N-terminal deletions of the GLP-1R revealed that the HRASP, not the SP itself, is essential for cell surface expression of the GLP-1R (ELISA 1.2%, p<0.001). Further, inhibition of GLP-1R glycosylation by tunicamycin treatment or an N-terminus deletion or mutation of all three glycosylation sites (Asn63,Asn82,Asn115) prevented receptor cell surface expression (ELISA 1.9%, p<0.001). Mutation of the conserved residues Trp39, Tyr69 and Tyr88 also significantly reduced cell surface expression (ELISA 25.1%, 3.7%, 2.3%, p<0.001) of GLP-1R independent of the SP cleavage or N-linked glycosylation.

Conclusion and Significance:

The N-terminus of GLP-1R regulates receptor trafficking and maturation. This study provides insight into the role of GLP-1R N-terminus on the receptor cell surface expression.

Acknowledgement:

The work in VK’s laboratory was funded by grants from BBSRC, MRC. AT is recipient of BBSRC DTG CASE studentship (Novo Nordisk is the industrial partner for the studentship).

(1) Gallwitz B, Pediatr Nephrol 25:1207, 2010

(2) Kochl R et al, J Biol Chem, 227:16131, 2002

(3) Chen et al, Am J Physiol Endocrinol Metab, 299:E62, 2010

(4) Whitaker et al, Plos One, 7:e32675, 2012