Prostanoid Receptors Location of Ligand Binding Sites

Recent advances in molecular biology have meant that the critical regions of amino acids to confer high affinity ligand binding are now beginning to be determined. For example, the importance of arginine at position 329 in the seventh transmembrane domain of all EP receptors has been examined by point mutations in the rabbit EP3 receptor. Thus, mutation of this amino acid to alanine or glutamate abolished the binding of [3H]-PGE2, whereas the mutation of aspartate 388 was without effect on ligand binding, but altered signal transduction, such that the EP3 receptor agonist, sul-prostone, was without effect even at high concentrations (279). Similar results have been obtained using mouse receptors: mutation of the equivalent arginine 309 to glutamate/va-line produced a loss in ligand binding, whereas switching it to lysine produced higher affinity binding (280). These data suggest that the seventh transmembrane domain of prostaglandin receptors is involved in both ligand binding and signal transduction.

Taken together with evidence that suggests that modification of the carboxyl group on the a-chain of prostaglandins tend to reduce agonist potency (219), the above findings suggest that this conserved arginine could be involved in interacting with the a-chain of prostaglandin ligands. Indeed, it has been shown that ligands containing methyl esters at C-l tend to display lower binding affinities than those containing negatively charged hydroxy groups. An example is the comparison of ligand binding of misoprostol methyl ester and free acid to the EP3 receptor (157). Furthermore, the rabbit EP3 receptor displays a 370fold decrease in affinity for PGE, methyl ester over PGE2 (279). On the other hand, the same authors demonstrated that sulprostone, which contains a sulfonamide at C-l that is bulkier

Agonists

Agonists

I-BOP

Figure 6.12. The structures of some TP receptor-selective agonists.

than a carboxyl group but still carries a negative charge (pKa = 5.25 cf 5.19 for the carboxyl group of PGE2), exhibited a threefold higher affinity than PGE2. These data suggest that a negative charge on C-l is important for high affinity ligand binding to EP receptors. Chang and colleagues (281) have investigated the relative contribution of the conserved arginine with respect to hydrogen bonding and ionic interactions that may be involved in ligand binding. They mutated the arginine to non-charged but polar glutamine or asparagines, or the non-polar leucine, to see how these changes affected the binding of PGE2. The mutation to leucine decreased binding affinity by around 40-fold, whereas binding was almost unaffected by the other two mutations. On the basis of this, they have suggested that hydrogen bonding may be enough for high affinity ligand binding.

It is also interesting to speculate whether the cysteine residue in the second extracellular loop forms a disulphide bridge that is important for receptor conformation. In the rabbit, mutation of this cysteine 204 to the uncharged alanine did not affect PGE2 binding (279). This is in contrast to results reported in human TP receptors, where mutation of the equivalent cysteine and another in the first extracellular loop abolished ligand binding (282, 283). Also, as the TP receptor fails to display ligand binding after reduction with dithiothreitol (284), and in other rhodop-sin-like receptors equivalent cysteine residues form a disulphide bridge, it has been suggested that cysteine 105 and cysteine 183 may form a disulphide bond essential to ligand binding.

Studies with human TP receptors have also highlighted several regions that are thought

Antagonists

L655240
GR32191 (vapiprost)
BAYu3405

Figure 6.32. (Continued.)

to be important in ligand binding. For example, the mutation of tryptophan 299, in the seventh transmembrane domain, to leucine produces a receptor that can bind the TP receptor agonist, U46619, but not the antagonist, SQ29548 (285). Furthermore, mutation of the conserved arginine 295, also in the seventh transmembrane domain of the human TP receptor, causes a reduction in ligand binding (285). Likewise, the importance of cysteine residues in the human TP receptor has also been investigated by mutational studies,

Table 6.12 Potencies of Some TP Receptor Agonists and Antagonists

Agonists

Equieffective Concentration Relative to U46619(=l)

Refs.

EP171

0.008-0.03

172,273

SQ26655

0.14-0.47

172

i-bop

0.12-0.25

273,274

sta2

36

275

Antagonists

paa

AH19437

AH23848B

GR32191

L-655240

BAYu3405

172, 276, 277 104, 172 172, 273 172, 277 269, 277 278

which have stressed the importance of those residues in the first and second extracellular loops (283). It also seems that glycosylation of the TP receptor may be important for ligand binding: when two extracellular glycosylation sites are mutated, the resulting receptor displays a loss of ligand binding (286).

The use of chimeric receptors has also been employed to attempt to dissect out regions of receptors important in ligand binding. For example, Kobayashi and colleagues (287) used chimeric DP and IP receptors to study possible domains involved in DP and IP receptor ligand binding. In substituting different regions of the mouse IP receptor with equivalent regions from the mouse DP receptor, they suggested that the sixth and seventh transmembrane domains of the IP receptor confer specificity to IP receptor ligands and that the third transmembrane domain of the DP receptor confers the specific binding of PGD,. They suggested that the IP receptor recognizes both the side-chains and cyclopentane rings of the ligands it binds. PGI, has a unique a-chain structure, caused by the presence of an additional ring attached to the cyclopentyl ring present in all prostanoids. It has been suggested that the IP receptor can recognize PGE,, but not PGE2, because of the lack of the C-5-C-6 double bond, which allows it to mimic the configuration of the side-chain in I series prostaglan-dins. Likewise, the binding of I- and E-series, but not D- and F-series prostaglandins, was explained by the lack of cyclopentane ring recognition for the latter two prostaglandin series. Kedzie and co-workers (288) employed a similar approach, mutating EP, receptor residues common to EP2 and EP, receptors, but not IP receptors to those present in IP receptors. They showed that mutating the human EP2 receptor leucine 304 in the seventh transmembrane domain to tyrosine resulted in an increase in potency of iloprost of around 100 times in a CAMP-dependent reporter gene assay. Likewise, mutation of the critical arginine 302 resulted in a loss of potency for all prostatested. This may be consistent with the above assertion that amino acids in the sixth and seventh transmembrane domains are relevant in the binding of the a-chain of IP ligands. Hence it can be seen that some progress is being made into the regions of prostanoid receptors that confer high affinity ligand binding.

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