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Site-directed mutagenesis of the human 5-HT1B receptor The 5-HT1B receptor plays diverse roles in the brain and the periphery where it is associated particularly with arterial vasoconstriction and it is thought to be involved in variant angina (Kaumann & Levy, 2006) and pulmonary hypertension (Morecroft & MacLean, 1998). Also, the utility of selective 5-HT1B/1D agonists in migraine treatment can be limited by actions on coronary vessels (Cocks et al., 1993). A selective, peripherally-acting, 5-HT1B antagonist might therefore be useful in cardiovascular disease. Glen et al. (1995) have developed a 3 pharmacophore model of the receptor and, to better understand the receptor binding site, single amino acid substitutions have been made in the human 5-HT1B (h5-HT1B) receptor. Residues Phe185 and Phe330 have been mutated to elucidate the effects on binding of the antagonist radioligand [3H]GR125743 (GE Life Science Products, Amersham). Mutations were generated in pcDNA 3.1 vectors, using the QuickChangeTM Site-Directed Mutagenesis kit (Stratagene) and were expressed in CHO cells transfected by electroporation. Transfected cells were cultured in DMEM (Invitrogen, Paisley, Scotland) supplemented with 10% fetal bovine serum (Invitrogen), penicillin 100U/l (Sigma, Poole, Dorset), streptomycin 100mg/l (Sigma), and L-glutamine 2mM (Sigma) until confluence, when the membranes were harvested. Membranes were incubated at several protein concentrations for each receptor for 60min in buffer containing 8nM [3H]GR125743 (twice the Kd for wild-type h5-HT1B by saturation binding). The reaction stopped by filtration through GF/B filters. Nonspecific binding was determined by 10μM SB216641. Saturation data were analyzed by nonlinear regression using GraphPad Prism and differences between specific binding at mutant receptors by two-way analysis of variance. In the F185A receptor there was a significant (P<0.0001) reduction in specific binding to the membranes (235±13pmol observed in WT to 183±6pmol; all values are at 200μg protein). This agrees with the findings of Granas et al. (2001). Greater reduction (to 58±3pmol; P<0.0001) occurred with mutation to the polar Tyr (F185Y). In contrast, mutation of Phe330 to either non-polar Ala (F330A; 15.4±0.3pmol) or polar Tyr (F330Y; 14.2±2.1), resulted very low levels of binding (P<0.0001). The large reduction in binding seen in F330Y is consistent with our modelling of the receptor, which suggests there is a face-to-edge interaction between the ligand and F330 via the para-hydrogen that is specifically eliminated by the mutation. Cocks TM et al. (1993) Br J Pharmacol 110, 360−368. |
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