The N-terminal region of the human 5-HT2C receptor has as a cleavable signal peptide

Abstract

The 5-hydroxytryptamine 2C (5-HT(2C)) receptor has a single nucleotide polymorphism (SNP) site at amino acid position 23 in its N-terminal tail. The polymorphism involves conversion of a cysteine to serine. The site, designated C23S, is located within a 32 amino acid long predicted signal peptide. The aim of the present study was to investigate whether the 5-HT(2C) receptor indeed has a functional cleavable signal peptide. For this purpose, ten N-terminally modified 5-HT(2C) receptors were constructed. Modifications included addition of the influenza virus hemagglutinin signal peptide, addition of a FLAG epitope, truncation of the N-terminal tail, and combinations of these changes. The receptors were transiently expressed in COS-7 cells. The relative amounts of receptors expressed at the membranes were quantified by [(3)H]-mesulergine radioligand binding. In one of the receptor constructs the FLAG epitope was inserted just after the endogenous putative signal peptide. Immunostaining with the M1 antibody, which recognizes the FLAG epitope only as free N-terminal entity, was used to detect whether the putative signal peptide preceding the FLAG epitope was cleaved off. The results suggest the following conclusions. The predicted signal peptide in the N-terminal tail of the 5-HT(2C) receptor acts as a cleavable signal peptide. Cleaving of the signal peptide is important for translocation of the wild type receptor to the plasma membrane. The two amino acids differentially encoded by the C23S SNP are likely absent from the mature 5-HT(2C) receptor.

A Putatively Functional Polymorphism in the HTR2C Gene is Associated with Depressive Symptoms in White Females Reporting Significant Life Stress

“…There is evidence that the Ser23 C allele is constitutively more active than the Cys23 G allele, with allele-specific analysis of the receptor showing functional differences in the activity of the Ser23 C minor allele relative to the Cys23 G allele [3] . Some studies, however, have failed to find in vitro differences between Ser23 and Cys23 variants [4] ; thus it is possible that the N-terminal region of the receptor containing this residue is not part of the mature protein in vivo [5] . The frequency of the Ser23 C allele is approximately 17% in unrelated Whites ( http://www.1000genomes.org/ ).…”

Tritium-labelled alkaloids: Synthesis and applications

“…Likely, more synthetic analogues than naturally occurring ergot alkaloids have been tritiated and used in various biological investigations. An example of this is the selective serotonin 2 receptor radioligand [ 3 H]mesulergine, used in several hundred published studies alone . Also, the use of [ 3 H]dihydroergocryptine as a probe for dopamine receptors demonstrates that tritiated dihydroanalogues can often serve as valuable proxy radioligands for the parent olefin .…”

“…An example of this is the selective serotonin 2 receptor radioligand [ 3 H]mesulergine, used in several hundred published studies alone. 28,29 Also, the use of [ 3 H]dihydroergocryptine as a probe for dopamine receptors demonstrates that tritiated dihydroanalogues can often serve as valuable proxy radioligands for the parent olefin. 30 However, in the case of indole alkaloid strychnine, we discovered that such a dihydroderivative for parent compound substitution was, surprisingly, not successful.…”

“…147 Nearly 200 publications have appeared using this radioligand to explore voltage-gated sodium channels. 148,149 The steroid-derived alkaloid tomatidine (29) was tritium labelled in a sequence of steps involving first oxidation of a 3-position alcohol to the ketone tomatidone. Next, a general exchange of tomatidone using tritiated water introduced tritium into the flanking 2-and 4-positions.…”

Association of Polymorphisms of Serotonin Transporter (5HTTLPR) and 5-HT2C Receptor Genes with Criminal Behavior in Russian Criminal Offenders

“…However, Okada et al [64] found that the Ser23 variant appears to be constitutively more active than Cys23. Jahnsen and Uhlèn [65] localized the C23S site within a cleavable signal peptide of the synthetized receptor protein. Cleaving of the signal peptide is important for translocation of the wild-type receptor to the plasma membrane, but they concluded that the site is probably absent from the mature HTR2C.…”