Solution Structure of the Second Extracellular Loop of Human Thromboxane A₂ Receptor

Abstract

Thromboxane A(2) receptor (TP receptor), a prostanoid receptor, belongs to the G protein-coupled receptor family, composed of three intracellular loops and three extracellular loops connecting seven transmembrane helices. The highly conserved extracellular domains of the prostanoid receptors were found in the second extracellular loop (eLP(2)), which was proposed to be involved in ligand recognition. The 3D structure of the eLP(2) would help to further explain the ligand binding mechanism. Analysis of the human TP receptor model generated from molecular modeling based on bacteriorhodopsin crystallographic structure indicated that about 12-14 A separates the N- and C-termini of the extra- and intracellular loops. Synthetic loop peptides whose termini are constrained to this separation are presumably more likely to mimic the native loop structure than the corresponding loop region peptide with unrestricted ends. To test this new concept, a peptide corresponding to the eLP(2) (residues 173-193) of the TP receptor has been made with the N- and C-termini connected by a homocysteine disulfide bond. Through 2D nuclear magnetic resonance (NMR) experiments, complete (1)H NMR assignments, and structural construction, the overall 3D structure of the peptide was determined. The structure shows two beta-turns at residues 180 and 185. The distance between the N- and C-termini of the peptide shown in the NMR structure is 14.2 A, which matched the distance (14.5 A) between the two transmembrane helices connecting the eLP(2) in the TP receptor model. This suggests that the approach using the constrained loop peptides greatly increases the likelihood of solving the whole 3D structures of the extra- and the intracellular domains of the TP receptor. This approach may also be useful in structural studies of the extramembrane loops of other G protein-coupled receptors.

Identification of Residues Important for Ligand Binding of Thromboxane A2 Receptor in the Second Extracellular Loop Using the NMR Experiment-guided Mutagenesis Approach

“…The synthetic constrained eLP2 peptide of the TP receptor has been shown to change the conformation upon the addition of the receptor antagonist SQ29,548 using fluorescence spectroscopic studies (1). The interaction was also supported by the separated CD spectroscopic studies (1). These results provide the evidences that the second extracellular loop of the TP receptor is involved in the receptor ligand recognition.…”

“…It is also believed that the initial contact residues on the molecular surface shall be specific. This hypothesis comes from our previous study (1). The synthetic constrained eLP2 peptide of the TP receptor has been shown to change the conformation upon the addition of the receptor antagonist SQ29,548 using fluorescence spectroscopic studies (1).…”

“…Thus, structural characterization of the extracellular functional domains of prostanoid receptors could help in understanding the specificities of ligand binding. In our current study, the structure of the highly conserved eLP2 has been characterized by high resolution NMR using a synthetic eLP2 peptide with constrained loop ends (1). To identify which residues make up the ligand recognition site of the receptor, SQ29,548 was added to the peptide to determine the interaction using high resolution two-dimensional 1 H NMR technique.…”

Mapping of a Ligand-binding Site for the Human Thromboxane A2 Receptor Protein

“…Furthermore, work done on the bradykinin B2 receptor using site-specific antibodies has identified the N-terminal portion of ED3 as crucial to both ligand binding and agonist function (30). More recently a constrained peptide containing amino acids representing ED3 of the TP receptor was shown to change conformation in response to the TP receptor antagonist SQ29,548 (18). These results in combination with the present data suggest that extracellular regions of seven transmembrane receptors may represent important ligand coordination sites.…”

“…To date, the information available concerning the TP receptor ligand-binding domain(s) has been mostly limited to mutational analyses using receptor chimeras or expressed receptor protein containing site-specific mutations as well as ligand interactions with modified receptor peptides or molecular modeling. Collectively results from these studies have implicated transmembrane domains I, III, IV, V, VI, and VII as well as extracellular domains II and III as potential regions for ligand coordination sites (11)(12)(13)(14)(15)(16)(17)(18). Since all of the cited receptor regions would not be expected to participate in or form the ligandbinding domain, it would seem that the interpretation of some of these results may be limited by the potential for gross alterations in receptor tertiary structure.…”