Biochemical and pharmacological study of N‐linked glycosylation of the human serotonin 5‐HT_7(a) receptor

Abstract

The 5-hydroxytryptamine (5-HT) 7(a) receptor is a G-protein-coupled receptor critically involved in human psychiatric and neurological disorders. In the present study, we evaluate the presence and the functional role of N-glycosylation of the human 5-HT 7 receptor. Western blot analysis of HEK293T cells transiently expressing the 5-HT 7(a) receptor in the presence of tunicamycin gave rise to a band shift, indicating the existence of an N-glycosylated form of the 5-HT 7(a) receptor. To further investigate this, we mutated the two predicted N-glycosylation sites (N5Q and N66Q) and compared the molecular mass of the immunoreactive bands with those of the wild-type receptor, indicating that both asparagines were N-glycosylated. The mutant receptors had the same binding affinity for [ 3 H]5-CT and the same potency and efficacy with regard to 5-HT-induced activation of adenylyl cyclase. However, there was a reduction in maximal ligand binding for the single and double mutants compared to the wild-type receptor. Next, membrane labelling and immunocytochemical studies demonstrated that the N-glycosylation mutants were expressed at the cell surface. We conclude that N-glycosylation is not important for cell surface expression of the 5-HT 7 receptor.

A Novel Interacting Protein SERP1 Regulates the N‐Linked Glycosylation and Function of GLP‐1 Receptor in the Liver

“…Accumulation of non‐glycosylated proteins triggers the unfolded protein response (UPR), which leads to cell death primarily through apoptosis [Gellynck et al, ]. Several UPR pathways are involved in apoptosis and autophagy in response to ER stress [Hauptmann et al, ; Zhang and Kaufman, ].…”

Development of a 5-HT7 receptor antibody for the rat: the good, the bad, and the ugly

“…For example, post translational modification of the 5-HT receptor is supported by several studies. Specifically, the 5-HT 7 receptor undergoes N-glycoslyation (Gellynck et al 2012 ) and C-terminal palmitoylation (Kvachina et al 2009 ). We have not tested whether a glycosylase would narrow the width of the bands observed.…”

Pharmacology and Therapeutic Potential of the 5-HT7 Receptor

“…Post-translational modifications of 5-HT 7 such as N-glycosylation of the N-terminus and palmitoylation of the C-terminus have been documented. N-Glycosylation does not appear to influence receptor activity, but reduced receptor expression was observed when possible glycosylation sites were mutated . Conversely, palmitoylation of the C-terminus has an impact on 5-HT 7 ’s constitutive activity .…”

“…N-Glycosylation does not appear to influence receptor activity, but reduced receptor expression was observed when possible glycosylation sites were mutated. 60 Conversely, palmitoylation of the C-terminus has an impact on 5-HT 7 's constitutive activity. 61 Mutation analysis indicated that Cys 404 and 438/441 are palmitoylated via a thioester bond in an agonist-dependent manner.…”