Abstract
A non-synonymous single nucleotide polymorphism of the human serotonin 5-HT2C receptor (5-HT2CR) gene that converts a cysteine to a serine at amino acid codon 23 (Cys23Ser) appears to impact 5-HT2CR pharmacology at a cellular and systems level. We hypothesized that the Cys23Ser alters 5-HT2CR intracellular signaling via changes in subcellular localization in vitro. Using cell lines stably expressing the wild-type Cys23 or the Ser23 variant, we show that 5-HT evokes intracellular calcium release with decreased potency and peak response in the Ser23 versus the Cys23 cell lines. Biochemical analyses demonstrated lower Ser23 5-HT2CR plasma membrane localization versus the Cys23 5-HT2CR. Subcellular localization studies demonstrated O-linked glycosylation of the Ser23 variant, but not the wild-type Cys23, may be a post-translational mechanism which alters its localization within the Golgi apparatus. Further, both the Cys23 and Ser23 5-HT2CR are present in the recycling pathway with the Ser23 variant having decreased colocalization with the early endosome versus the Cys23 allele. Agonism of the 5-HT2CR causes the Ser23 variant to exit the recycling pathway with no effect on the Cys23 allele. Taken together, the Ser23 variant exhibits a distinct pharmacological and subcellular localization profile versus the wild-type Cys23 allele, which could impact aspects of receptor pharmacology in individuals expressing the Cys23Ser SNP.
Highlights
Single nucleotide polymorphisms (SNPs) of the human 5-HT2CR gene (HTR2C), located on the X chromosome, associate with behavioral phenotypes, psychiatric conditions, and response to psychiatric medications including atypical antipsychotics and antidepressants[15,16,17,18,19,20,21,22]
We employed RNAseq analyses to demonstrate that Chinese Hamster Ovary (CHO) cell lines express some of the major players in 5-HT2CR localization and signaling, including Camk[1] (Calmodulin)[38,39], Pten (PTEN, phosphatase and tensin homolog)[40,41], and low levels of Dlg[4] (PSD95, postsynaptic density 95)[32]
We investigated colocalization of the 5-HT2CR variants within the Golgi apparatus due to the presence of GalNac transferase and O-glycosylation in the Golgi apparatus[54,55,56,57,58,59] and the knowledge that O-glycosylation impacts protein folding and structure as well as possibly localization and targeting of proteins[61,62,63] to support the hypothesis that O-glycosylation could be a part of protein quality control for the 5-HT2CR and, impacted by the Cys23Ser SNP
Summary
Single nucleotide polymorphisms (SNPs) of the human 5-HT2CR gene (HTR2C), located on the X chromosome, associate with behavioral phenotypes, psychiatric conditions, and response to psychiatric medications including atypical antipsychotics and antidepressants[15,16,17,18,19,20,21,22]. The Cys23Ser SNP may impact phenotypic behaviors and cellular function through alterations in the structural integrity of the 5-HT2CR protein, the efficiency of 5-HT2CR ligands and signal transduction mechanisms and/or receptor subcellular localization profiles[30]. Following agonist-mediated receptor endocytosis, the 5-HT2CR can be resensitized and sent back to the plasma membrane from the early endosomes or recycling endosomes[32,35,36,37] These pathways are integral steps in GPCR function, the actual impact of the Cys23Ser SNP on 5-HT2CR subcellular localization, at the plasma membrane, is unknown. ++ i release, immunocytochemistry, WesTM automated immunoblotting, radioligand binding, surface biotinylation) to demonstrate that the Ser[23] variant attenuates agonist-induced intracellular signaling and basally has lower plasma membrane expression with a distinct localization pattern within the recycling pathway than the wild-type Cys[23]
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