title: Post-translational modifications of the serotonin type 4 receptor heterologously expressed in mouse rod cells.
abstract: G-protein-coupled serotonin receptor type 4 (5-HT(4)R) is a pharmacological target implicated in a variety of gastrointestinal and nervous system disorders. As for many other integral membrane proteins, structural and functional studies of this receptor could be facilitated by its heterologous overexpression in eukaryotic systems that can perform appropriate post-translational modifications (PTMs) on the protein. We previously reported the development of an expression system that employs rhodopsin's biosynthetic machinery in rod cells of the retina to express heterologous G-protein-coupled receptors (GPCRs) in a pharmacologically functional form. In this study, we analyzed the glycosylation, phosphorylation, and palmitoylation of 5-HT(4)R heterologously expressed in rod cells of transgenic mice. We found that the glycosylation pattern in 5-HT(4)R was more complex than in murine and bovine rhodopsin. Moreover, overexpression of this exogenous GPCR in rod cells also affected the glycosylation pattern of coexisting native rhodopsin. These results highlight not only the occurrence of heterogeneous PTMs on transgenic proteins but also the complications that non-native PTMs can cause in the structural and functional characterization of both endogenous and heterologous protein targets.
authors.affiliation: Polgenix Inc., Cleveland, Ohio 44106, United States.
authors.forename: David
authors.initials: D
authors.lastname: Salom
authors.forename: Benlian
authors.initials: B
authors.lastname: Wang
authors.forename: Zhiqian
authors.initials: Z
authors.lastname: Dong
authors.forename: Wenyu
authors.initials: W
authors.lastname: Sun
authors.forename: Pius
authors.initials: P
authors.lastname: Padayatti
authors.forename: Steven
authors.initials: S
authors.lastname: Jordan
authors.forename: John A
authors.initials: JA
authors.lastname: Salon
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Palczewski
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D005260:Female; D006031:Glycosylation; D054878:Lipoylation; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D041681:NIH 3T3 Cells; D010766:Phosphorylation / Q000235:genetics; D011499:Protein Processing, Post-Translational / Q000235:genetics*; D021381:Protein Transport / Q000235:genetics; D044422:Receptors, Serotonin, 5-HT4 / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D017948:Retinal Rod Photoreceptor Cells / Q000378:metabolism*; D012243:Rhodopsin / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism
chemical_list: C504672:Rrh protein, mouse; D044422:Receptors, Serotonin, 5-HT4; D012243:Rhodopsin