Cloning and expression of cDNA for a human thromboxane A2 receptor

Abstract

Thromboxane A2 is a very unstable arachidonate metabolite, yet a potent stimulator of platelet aggregation and a constrictor of vascular and respiratory smooth muscles. It has been implicated as a mediator in diseases such as myocardial infarction, stroke and bronchial asthma. Using a stable analogue of this compound we recently purified the human platelet thromboxane A2 receptor to apparent homogeneity. Using an oligonucleotide probe corresponding to its partial amino-acid sequence, we have obtained a complementary DNA clone encoding this receptor from human placenta and a partial clone from cultured human megakaryocytic leukaemia cells. The placenta cDNA encodes a protein of 343 amino acids with seven putative transmembrane domains. The protein expressed in COS-7 cells binds drugs with affinities identical to those of the platelet receptor, and that in Xenopus oocytes opens Ca2(+)-activated Cl- channel on agonist stimulation. Northern blot analysis and nucleotide sequences of the two clones suggest that an identical species of the thromboxane A2 receptor is present in platelets and vascular tissues. This first report on the molecular structure of an eicosanoid receptor will promote the molecular pharmacology and pathophysiology of these bioactive compounds.

Thromboxane A2 Signalling in Humans: a ‘Tail’ of Two Receptors

“…A cDNA for the human TXA 2 receptor (TP) was originally cloned from placenta and the platelet like MEG-01 cell line [20] and since then cDNAs for TPs from a number of species have been cloned and characterised [21][22][23][24][25]. All TPs are predicted to share the seven α-helical transmembrane domain arrangement typical of other members of the GPCR superfamily [26].…”

“…Despite this, Raychowdhury et al, [32] later isolated a cDNA encoding a second form (isoform) of the TP from a human umbilical vein endothelial cell (HUVEC) cDNA library. Thus, in humans, there are 2 receptors for TXA 2 , termed TPα and TPβ [20,31,32], where the TP originally cloned from platelet/placenta is commonly referred to as the TPα isoform and that from HUVECs referred to as the TPβ isoform. The TP isoforms are identical for their N-terminal 328 amino acid (aa) residues but differ exclusively in their carboxyl terminal cytoplasmic (C-tail) domains such that TPα has 15 amino acid residues within its unique C-tail sequence and TPβ has 79 residues within its C-tail sequence [20,31,32; Figure 1].…”

“…Thus, in humans, there are 2 receptors for TXA 2 , termed TPα and TPβ [20,31,32], where the TP originally cloned from platelet/placenta is commonly referred to as the TPα isoform and that from HUVECs referred to as the TPβ isoform. The TP isoforms are identical for their N-terminal 328 amino acid (aa) residues but differ exclusively in their carboxyl terminal cytoplasmic (C-tail) domains such that TPα has 15 amino acid residues within its unique C-tail sequence and TPβ has 79 residues within its C-tail sequence [20,31,32; Figure 1]. TPα and TPβ are encoded by the single TP gene, on chromosome 19p13.3 [31], and arise by a novel differential splicing mechanism within Exon 3 whereby nucleotides 984 -1642 of the TPα mRNA behave as an Intron (Intron 2b) within the TPβ mRNA [20,31,32; Figure 1].…”

“…The TP isoforms are identical for their N-terminal 328 amino acid (aa) residues but differ exclusively in their carboxyl terminal cytoplasmic (C-tail) domains such that TPα has 15 amino acid residues within its unique C-tail sequence and TPβ has 79 residues within its C-tail sequence [20,31,32; Figure 1]. TPα and TPβ are encoded by the single TP gene, on chromosome 19p13.3 [31], and arise by a novel differential splicing mechanism within Exon 3 whereby nucleotides 984 -1642 of the TPα mRNA behave as an Intron (Intron 2b) within the TPβ mRNA [20,31,32; Figure 1]. Reverse transcriptase polymerase chain reaction (RT PCR) experiments indicated that HUVECs express only TPβ [32] whereas human platelets were reported to express both TPα and TPβ isoforms [33].…”

TBXA2R gene variants associated with bleeding

“…The human TPα receptor was the first human eicosanoid receptor cloned and is a typical Class A rhodopsin-like G protein-coupled receptor (GPCR) (1) with sequence variants in TBXA2R , which encodes the TPα receptor implicated in asthma, atopic dermatitis, and, of particular relevance to this review, an autosomal dominant bleeding disorder (2). …”