title: 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.
authors.affiliation: Department of Oral Biology, University of Manitoba, Winnipeg, Manitoba, Canada.
authors.forename: Raja
authors.initials: R
authors.lastname: Chakraborty
authors.forename: Sai Prasad
authors.initials: SP
authors.lastname: Pydi
authors.forename: Scott
authors.initials: S
authors.lastname: Gleim
authors.forename: Shyamala
authors.initials: S
authors.lastname: Dakshinamurti
authors.forename: John
authors.initials: J
authors.lastname: Hwa
authors.forename: Prashen
authors.initials: P
authors.lastname: Chelikani
mesh_terms: D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid / Q000378:metabolism / Q000494:pharmacology; D000409:Alanine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive; D019086:Bridged Bicyclo Compounds, Heterocyclic; D019556:COS Cells; D002118:Calcium / Q000378:metabolism; D002522:Chlorocebus aethiops; D005231:Fatty Acids, Unsaturated; D005998:Glycine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D006834:Hydrazines / Q000378:metabolism / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000819:agonists / Q000037:antagonists & inhibitors / Q000378:metabolism; D020125:Mutation, Missense; D020641:Polymorphism, Single Nucleotide; D017433:Protein Structure, Secondary; D011869:Radioligand Assay; D044244:Receptors, Thromboxane A2, Prostaglandin H2 / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D013329:Structure-Activity Relationship; D013696:Temperature
chemical_list: D019086:Bridged Bicyclo Compounds, Heterocyclic; D005231:Fatty Acids, Unsaturated; D006834:Hydrazines; D050505:Mutant Proteins; D044244:Receptors, Thromboxane A2, Prostaglandin H2; D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; C045749:SQ 29548; D000409:Alanine; D002118:Calcium; D005998:Glycine