Identification of Domains Conferring Ligand Binding Specificity to the Prostanoid Receptor

Abstract

To identify domains conferring ligand binding specificity to prostanoid receptors, we constructed a series of chimeric receptors by successively replacing the regions from the carboxyl-terminal tail of mouse prostacyclin (prostaglandin I (PGI)) receptor (mIP) with the corresponding regions of the mouse PGD receptor (mDP showed no affinity for the mIP, mDP, and all the chimeric receptors. These results suggest that the sixth to seventh transmembrane domain of the mIP receptor confers the specificity of this receptor to bind selectively to PGE 1 and not to PGE 2 and that the third transmembrane domain of the mDP receptor confers the selective binding of PGD 2 to this receptor. Prostaglandins (PGs)1 contain prostanoic acid as a central structural element. PGs have two structural features in the prostanoic acid framework. First, they have functional groups on the cyclopentane ring, which classifies them into four types, D, E, F, and I. Second, they are classified into three series, 1, 2, and 3, by the number of double bonds in the side chains. Additionally, another cyclooxygenase product, thromboxane A 2 has an oxane ring instead of the cyclopentane ring. These prostanoids act on eight types and subtypes of the receptors. They are the PGD receptor (DP), the EP 1 , EP 2 , EP 3 , and EP 4 subtypes of the PGE receptor, the PGF receptor (FP), the PGI receptor (IP), and the thromboxane A 2 receptor (TP) (1-4). These receptors can recognize the structural differences of prostanoid molecules. The binding affinities of these receptors to prostanoid molecules are determined primarily by the cyclopentane ring structures of ligands. For example, the DP receptor shows the highest affinities to PGD 2 and PGD 1 , but affinities to other prostanoids are at least 2 orders of magnitude less. One exception is the IP receptor, which shows the affinity to PGE 1 almost comparable to PGI analogs such as iloprost. This receptor, however, can bind PGE 2 with much lower affinity, suggesting that the IP receptor can discriminate a difference in the side chains.We have cloned cDNAs for all of these types and subtypes of the mouse prostanoid receptors (5-13). These studies revealed that the prostanoid receptors belong to the G protein-coupled rhodopsin type receptor superfamily. They have several regions conserved specifically among them. These conserved regions may participate in the construction of binding domains for structures common to prostanoid molecules, whereas the other regions may confer specificity for ligand binding. For example, the arginine in the seventh transmembrane domain, which is conserved in all of the prostanoid receptors, was proposed to be the binding site for the carboxyl group of prostanoid molecules (5, 14, 15). In fact, Funk et al. (16) have shown that a point mutation at this arginine residue in the human TP receptor results in loss of ligand binding activity. However, structural domains of the prostanoid receptors conferring specificity for ligand binding are as yet unknown.

Molecular Basis for Agonist Selectivity and Activation of the Orphan Bombesin Receptor Subtype 3 Receptor

“…Likewise, with CCK B R, both EC2 and EC3 were important for the CCK affinity (Silvente-Poirot et al, 1998). In contrast, only the TM3 region of the mouse PGD receptor was essential for prostaglandin D 2 binding (Kobayashi et al, 1997). These results demonstrate the marked variability in molecular determinants not only for different unrelated receptors but also for closely related receptors for peptide agonists.…”

Identification of Determinants of Ligand Binding Affinity and Selectivity in the Prostaglandin D2 Receptor CRTH2

“…The data presented here support this hypothesis and suggest that a similar interaction between Lys-209 and the carboxylate of PGD 2 may occur. This would further differentiate the CRTH2 receptor from other prostanoid receptors, for which it has been suggested that TM VI and VII are responsible for recognition of the prostaglandin ␣-chain and terminal carboxylate by the DP and IP receptors (37) and that a conserved arginine in TM VII in the TP (38) and EP3 receptors (23,39) Interactions between the receptor and cyclopentyl ring substituents of prostaglandin ligands are thought to determine the ligand selectivity of each receptor. For the DP receptor, residues in TM I and II have been demonstrated to confer high affinity and selectivity of PGD 2 binding, suggesting that the cyclopentyl ring of PGD 2 is positioned next to TM I and II (32).…”

Tyrosine 220 in the 5th Transmembrane Domain of the Neuromedin B Receptor Is Critical for the High Selectivity of the Peptoid Antagonist PD168368

“…However, with peptide agonists for the B 2 bradykinin receptor (63), the corticotropin-releasing factor receptor (69), or -and ␦-opioid receptors (70), the transmembrane regions are not important. In similar fashion, several nonpeptide agonists have been shown to primarily interact with GPCR upper transmembrane regions including nonpeptide agonists for adrenergic receptors (54,55), muscarinic cholinergic receptors (56,71,72), or eicosanoid (73,74). High affinity receptor binding of some peptide antagonists also have been shown to depend primarily on interaction with the upper transmembrane domains of receptors such as the interaction of NPC567 and Hoe140 with the B 2 bradykinin receptor (57,58) or BQ-123 with the ET A -R (13), whereas with other GPCRs such as interaction of CCK-JMV179 with the CCK-A receptor (12), the upper transmembrane regions are not important.…”