We describe the cloning and characterization of a human 5‐HT6 serotonin receptor. The open reading frame is interrupted by two introns in positions corresponding to the third cytoplasmic loop and the third extracellular loop. The human 5‐HT6 cDNA encodes a 440‐amino‐acid polypeptide whose sequence diverges significantly from that published for the rat 5‐HT6 receptor. Resequencing of the rat cDNA revealed a sequencing error producing a frame shift within the open reading frame. The human 5‐HT6 amino acid sequence is 89% similar to the corrected rat sequence. The recombinant human 5‐HT6 receptor is positively coupled to adenylyl cyclase and has pharmacological properties similar to the rat receptor with high affinity for several typical and atypical antipsychotics, including clozapine. The receptor is expressed in several human brain regions, most prominently in the caudate nucleus. The gene for the receptor maps to the human chromosome region 1p35–p36. This localization overlaps that established for the serotonin 5‐HT1Dα receptor, suggesting that these may be closely linked. Comparison of genomic and cDNA clones for the human 5‐HT6 receptor also reveals an RsaI restriction fragment length polymorphism within the coding region.
Several models of agonist binding to G protein-coupled 5-hydroxytryptamine [5-HT] (serotonin) receptors have highlighted the potential importance of highly conserved aromatic residues for ligand binding and agonist efficacy. In this study, we tested these models by constructing and characterizing a number of point mutations of conserved and nonconserved aromatic residues using the 5-HT2A receptor as a model system. Mutations of three highly conserved tryptophans (W200A, W336A, and W367A) proposed to reside near the binding pocket markedly reduced agonist affinity and efficacy at 5-HT2A receptors. Mutations of two other highly conserved aromatic residues postulated to be near the agonist binding site (F340L and Y370A) also had dramatic effects on agonist binding and efficacy. Point mutations of neighboring conserved phenylalanines (F339L and F365L) had minimal effects on agonist binding, although the F365L mutation diminished agonist efficacy. Finally, mutations of two nonconserved aromatic residues (F125L and F383A) not predicted to be near the binding pocket had no effects on agonist binding, potency, or efficacy. Our results are best explained by models that suggest that helices III, V, VI, and VII can form a unit of interacting helices in which highly conserved aromatic residues are oriented toward the center of the helical aggregate to form an aromatic pocket. In addition, our novel results identify a series of aromatic residues essential for agonist-induced second messenger production. These results demonstrate that highly conserved aromatic residues residing in neighboring helices provide the optimum environment for both agonist binding and activation of 5-HT2A receptors.
Background SARS-CoV-2 variants of concern (VOCs) have threatened COVID-19 vaccine effectiveness. We aimed to assess the effectiveness of the ChAdOx1 nCoV-19 vaccine, predominantly against the delta (B.1.617.2) variant, in addition to the cellular immune response to vaccination. Methods We did a test-negative, case-control study at two medical research centres in Faridabad, India. All individuals who had a positive RT-PCR test for SARS-CoV-2 infection between April 1, 2021, and May 31, 2021, were included as cases and individuals who had a negative RT-PCR test were included as controls after matching with cases on calendar week of RT-PCR test. The primary outcome was effectiveness of complete vaccination with the ChAdOx1 nCoV-19 vaccine against laboratory-confirmed SARS-CoV-2 infection. The secondary outcomes were effectiveness of a single dose against SARS-CoV-2 infection and effectiveness of a single dose and complete vaccination against moderate-tosevere disease among infected individuals. Additionally, we tested in-vitro live-virus neutralisation and T-cell immune responses to the spike protein of the wild-type SARS-CoV-2 and VOCs among healthy (anti-nucleocapsid antibody negative) recipients of the ChAdOx1 nCoV-19 vaccine. Findings Of 2379 cases of confirmed SARS-CoV-2 infection, 85 (3•6%) were fully vaccinated compared with 168 (8•5%) of 1981 controls (adjusted OR [aOR] 0•37 [95% CI 0•28-0•48]), giving a vaccine effectiveness against SARS-CoV-2 infection of 63•1% (95% CI 51•5-72•1). 157 (6•4%) of 2451 of cases and 181 (9•1%) of 1994) controls had received a single dose of the ChAdOx1 nCoV-19 vaccine (aOR 0•54 [95% CI 0•42-0•68]), thus vaccine effectiveness of a single dose against SARS-CoV-2 infection was 46•2% (95% CI 31•6-57•7). One of 84 cases with moderate-to-severe COVID-19 was fully vaccinated compared with 84 of 2295 cases with mild COVID-19 (aOR 0•19 [95% CI 0•01-0•90]), giving a vaccine effectiveness of complete vaccination against moderate-to-severe disease of 81•5% (95% CI 9•9-99•0). The effectiveness of a single dose against moderate-to-severe disease was 79•2% (95% CI 46•1-94•0); four of 87 individuals with moderate-to-severe COVID-19 had received a single dose compared with 153 of 2364 participants with mild disease (aOR 0•20 [95% CI 0•06-0•54]). Among 49 healthy, fully vaccinated individuals, neutralising antibody responses were lower against the alpha (B.1.1.7; geometric mean titre 244•7 [95% CI 151•8-394•4]), beta (B.1.351; 97•6 [61•2-155•8]), kappa (B.1.617.1; 112•8 [72•7-175•0]), and delta (88•4 [61•2-127•8]) variants than against wild-type SARS-CoV-2 (599•4 [376•9-953•2]). However, the antigen-specific CD4 and CD8 T-cell responses were conserved against both the delta variant and wild-type SARS-CoV-2. Interpretation The ChAdOx1 nCoV-19 vaccine remained effective against moderate-to-severe COVID-19, even during a surge that was dominated by the highly transmissible delta variant of SARS-CoV-2. Spike-specific T-cell responses were maintained against the delta variant. Such cellular immune p...
The mechanism by which antagonists down-regulate 5-HT2A receptors in unknown. We here report that a variety of 5-HT2A antagonists induce a change in the subcellular distribution of 5-HT2A receptors both in vitro and in vivo. In a stably transfected NIH 3T3 cell-line, brief exposure to 1 muM clozapine caused a 2.5-fold increase in intracellular 5-HT2A-like immunoreactivity, as measured by confocal microscopy. Confirmatory studies utilizing a biotin-trap technique, demonstrated that the increase in intracellular immunoreactivity results from internalization of receptor from the cell surface. Exposure of transfected cells to other 5-HT2A receptor antagonists produced similar increases in intracellular 5-HT2A-like immunoreactivity. In vivo administration of clozapine (20 mg/kg, sc, X 7 days) caused a greater than twofold increase in intracellular immunoreactivity in cell bodies of cortical pyramidal neurons. Additionally, chronic clozapine administration was associated with decrease in labeling of apical dendrites on pyramidal cells. These results show that clozapine causes a change in subcellular distribution of 5-HT2A receptors in vitro and in vivo.
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