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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.

SUBMITTER: Salom D

PROVIDER: S-EPMC3268512 | biostudies-literature | 2012 Jan

REPOSITORIES: biostudies-literature

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Post-translational modifications of the serotonin type 4 receptor heterologously expressed in mouse rod cells.

Salom David D Wang Benlian B Dong Zhiqian Z Sun Wenyu W Padayatti Pius P Jordan Steven S Salon John A JA Palczewski Krzysztof K

Biochemistry 20111220 1

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 rhodops ...[more]

PMID: 22145929

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Project description:The fracture resistance of cortical bone and matrix hydration are known to decline with advanced aging. However, the underlying mechanisms remain poorly understood, and so we investigated levels of matrix proteins and post-translational modifications (PTM) of collagen I in extracts from the tibia of 6-mo. and 20-mo. old BALB/c mice (female and male analysis done separately). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis revealed that the levels of collagen I deamidation at specific asparagine (Asn) and glutamine (Gln) residues significantly increased with age. Other non-enzymatic PTMs such as carboxymethylation of lysine (CML) were detected as well, but the relative abundance did not vary with age. No significant age-related differences in the abundance of hydroxylysine glycosylation sites were found, but hydroxylation levels at a few of the numerous lysine and proline hydroxylation sites significantly changed by a small amount with age. We performed molecular modeling and dynamics (MD) simulations for three triple helical fragments representing collagen I regions with prominent age-dependent increases in deamidation as identified by LC-MS/MS of male extracts. These 3 fragments included deamidated Asn and Gln residues as follows: 1) an Asn428 site of the α2(I) chain in which deamidation levels increased from 4.4% at 6-mo. to 8.1% at 20-mo., 2) an Asn983 site of the α2(I) chain with a deamidation increase from 18.3% to 36.8% with age and an Asn1052 site of the α1(I) chain with consistent deamidation levels of ~60% across the age groups, and 3) a Gln410 site of the α1(I) chain that went from no detectable deamidation at 6-mo. to 2.7% at 20-mo. and a neighboring Asn421 site of the same chain with an age-related deamidation increase from 3.6% to 16.3%. The deamidation levels at these sites inversely correlated with an estimate of toughness determined from three-point bending tests of the femur mid-diaphysis. MD revealed that the sidechains become more negatively charged at deamidated sites and that deamidation alters hydrogen bonding with water along the collagen backbone while increasing water interactions with the aspartic and glutamic acid sidechains. Our findings suggest a new mechanism of the age-dependent reduction in the fracture resistance of cortical bone whereby deamidation of Asn and Glu residues redistributes bound water within collagen I triple helix.

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Post-translational modifications of the serotonin type 4 receptor heterologously expressed in mouse rod cells.

Publications

Post-translational modifications of the serotonin type 4 receptor heterologously expressed in mouse rod cells.

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