Site-directed mutations and the polymorphic variant Ala160Thr in the human thromboxane receptor uncover a structural role for transmembrane helix 4

Abstract

The human thromboxane A2 receptor (TP), belongs to the prostanoid subfamily of Class A GPCRs and mediates vasoconstriction and promotes thrombosis on binding to thromboxane (TXA2). In Class A GPCRs, transmembrane (TM) helix 4 appears to be a hot spot for non-synonymous single nucleotide polymorphic (nsSNP) variants. Interestingly, A160T is a novel nsSNP variant with unknown structure and function. Additionally, within this helix in TP, Ala160(4.53) is highly conserved as is Gly164(4.57). Here we target Ala160(4.53) and Gly164(4.57) in the TP for detailed structure-function analysis. Amino acid replacements with smaller residues, A160S and G164A mutants, were tolerated, while bulkier beta-branched replacements, A160T and A160V showed a significant decrease in receptor expression (Bmax). The nsSNP variant A160T displayed significant agonist-independent activity (constitutive activity). Guided by molecular modeling, a series of compensatory mutations were made on TM3, in order to accommodate the bulkier replacements on TM4. The A160V/F115A double mutant showed a moderate increase in expression level compared to either A160V or F115A single mutants. Thermal activity assays showed decrease in receptor stability in the order, wild type>A160S>A160V>A160T>G164A, with G164A being the least stable. Our study reveals that Ala160(4.53) and Gly164(4.57) in the TP play critical structural roles in packing of TM3 and TM4 helices. Naturally occurring mutations in conjunction with site-directed replacements can serve as powerful tools in assessing the importance of regional helix-helix interactions.

Figure 1. Two-dimensional representation of the TPα amino acid sequence.

Amino acids are shown in single-letter codes. Shown are the seven transmembrane helices, the disulphide bond between the Cys102 and Cys183 (green colored residues), the N-glycosylated residues Asn4 and Asn16 (orange colored residues), and the rho-1D4 octapeptide epitope tag at the C-terminus. The two conserved residues Ala160^4.53 and Gly164^4.57 on TM4 along with the residues on TM3 mutated in this study are shown in red.

Figure 2. Saturation binding assays of wild type TP and the mutant receptors using the TP antagonist [³H] SQ 29,548.

Saturation assays with membrane bound TP and the mutant receptors, TM4 mutants in panel A and TM3 mutants in panel B, were performed with different concentrations of [³H] SQ 29,548. A one way ANOVA analysis without any post hoc test showed that except for G164V in TM4 there were no significant difference between TP and the mutants at significance level of p<0.05. The data is from a minimum of three independent experiments, with each point in duplicate.

Figure 3. Heterologous competition curves of TP and select mutants using the unlabeled agonist U46619 and antagonist [³H] SQ 29,548.

The Ki values (µM) are shown in parenthesis.

Figure 4. Characterization of Gαq-mediated signalling of the TP and mutant receptors.

The data shows agonist U46619 induced calcium release for TP, mutants and mock transfected (vector pMT4) HEK293T cells, and is expressed as a percentage of the TP activity. Ala160 mutants (panel A), Gly164 mutants and mock transfected cells (panel B), TM3 mutants (panel C) and double mutants (panel D). The results are from at least three independent experiments performed in duplicate.

Figure 5. Characterization of basal or agonist-independent activity.

Bar plot representation of the basal amount of calcium released by TP and mutants per picomole of functional protein expressed (see methods). The G164V mutant could not be assessed as it did not bind to the antagonist, and the amount of functional receptor could not be calculated. Results are obtained from a minimum of two independent experiments done in duplicate. The single astrix indicate there is a significant difference in the amount of calcium released at zero concentration of agonist with respect to wild type TP at significance level p<0.05. Error bars indicate mean ± SD.

Figure 6. Thermal sensitivity of wild type TP and select mutants.

It was measured in terms of the ability of the TP and mutants at positions 4.53 and 4.57 to retain antagonist binding after incubation at 25°C (panel A), 37°C (panel B) and 42°C (panel C) as a function of time. The receptor activity decreased in the order, TP>A160S>A160V>A160T>G164A, with G164A being the least stable. The results are mean ±SE and are from minimum of three independent experiments done in duplicate.

Figure 7. Molecular models of antagonist bound TP and G164V mutant and comparison with the crystal structures of rhodopsin and antagonist bound β₂-AR.

Panel A, Molecular model of TP bound SQ 29,548 superimposed with the structures of rhodopsin (PDB ID 1U19) and antagonist bound β₂-AR (PDB ID 2RH1). The two residues at positions 4.53 and 4.57 on TM4 in both rhodopsin and β₂-AR were previously studied. For structural comparison all the three structures were superimposed. The color coding is as follows; Ala164 and Ala168 in rhodopsin (red), Ala160 and Gly164 in TP (blue) and Ser161 and Ser164 in β₂-AR (green). Panel B, Molecular models of TP (yellow) and G164V (green) superimposed. The important amino acids Gly164, Val164 and Ser191 are shown. Notice that Ser191 loses interaction with the antagonist SQ 29,548 in the G164V mutant.

Figure 8. Molecular models of agonist bound TP and Ala160 mutants.

Wild type (panel A), A160S (panel B), A160T (panel C) and A160V (panel D), and the important amino acids are shown in each model.