Partial Agonism through a Zinc-Ion Switch Constructed between Transmembrane Domains III and VII in the Tachykinin NK₁Receptor

Abstract

Partly due to lack of detailed knowledge of the molecular recognition of ligands the structural basis for partial versus full agonism is not known. In the beta(2)-adrenergic receptor the agonist binding site has previously been structurally and functionally exchanged with an activating metal-ion site located between AspIII:08-or a His residue introduced at this position in transmembrane domain (TM)-III-and a Cys residue substituted for AsnVII:06 in TM-VII. Here, this interhelical, bidentate metal-ion site is without loss of Zn(2+) affinity transferred to the tachykinin NK(1) receptor. In contrast to the similarly mutated beta(2)-adrenergic receptor, signal transduction-i.e., inositol phosphate turnover-could be stimulated by both Zn(2+) and by the natural agonist, Substance P in the mutated NK(1) receptor. The metal-ion acted as a 25% partial agonist through binding to the bidentate zinc switch located exactly one helical turn below the two previously identified interaction points for Substance P in, respectively, TM-III and -VII. The metal-ion chelator, phenantroline, which in the beta(2)-adrenergic receptor increased both the potency and the agonistic efficacy of Zn(2+) or Cu(2+) in complex with the chelator, also bound to the metal-ion site-engineered NK(1) receptor, but here the metal-ion chelator complex instead acted as a pure antagonist. It is concluded that signaling of even distantly related rhodopsin-like 7TM receptors can be activated through Zn(2+) coordination between metal-ion binding residues located at positions III:08 and VII:06. It is suggested that only partial agonism is obtained through this simple well defined metal-ion coordination due to lack of proper interactions with residues also in TM-VI.

Construction of a High Affinity Zinc Binding Site in the Metabotropic Glutamate Receptor mGluR1

“…This indicates that the site constructed in K260H is relatively specific for Zn 2ϩ . The effect of Cu 2ϩ (as the free ion or complexed with phenanthroline) on metal ion sites constructed in TMs of family A GPCRs have varied from being just as potent to being significantly weaker than that of that of Zn 2ϩ (41,42,44). Whether Cu 2ϩ and Co 2ϩ do not bind to K260H or whether they bind but, unlike Zn 2ϩ , are unable to induce a conformational lock on the region is impossible to determine.…”

“…In both scenarios, Zn 2ϩ binding clearly has to involve residues on two strands in the ATD lip to have any implications for the signal transduction through the receptor. Analogously, both antagonistic and agonistic zinc sites created by mutations in one TM of family A and B GPCRs have been significantly "improved" by the introduction of coordinating zinc ligands in another TM (39,(42)(43)(44)46).…”

“…The typical coordination arrangement of Zn 2ϩ binding to proteins is tetrahedral or distorted tetrahedral, where the protein most often contributes with three or four ligands to the binding (27)(28)(29). However, zinc and other metal ions are able to coordinate to proteins in a bidentate manner, with solvent molecules acting as the remaining ligands (29, 50 -52), and several bidentate zinc sites have been introduced in the TMs of GPCRs (39,40,(42)(43)(44)(45)(46). Considering that the residues involved in zinc binding to K260H mGluR1 are located in the top of the ATD lobe, the region would be expected to be solvent-accessible.…”

High Affinity Agonistic Metal Ion Binding Sites within the Melanocortin 4 Receptor Illustrate Conformational Change of Transmembrane Region 3

“…The TM regions were reconstructed to avoid problems with structural features derived from specific amino acid sequences found in the rhodopsin receptor but not in the MC4R or vice versa. The resulting TM (1)(2)(3)(4)(5)(6)(7) 320 (including the postulated eight ␣-helix), respectively. The positions of the three first amino acids in the cytosolic part of the helices in the rhodopsin model were used to orientate the helical segments in relation to each other.…”

“…Such artificial intrahelical and interhelical binding sites have been used effectively to determine the orientation and exact distances between the ␣-helices of the tachykinin, opioid, and the ␤-adrenergic receptor families (4 -6). Moreover, in two previous studies (6,7), an interhelical binding site has been created that allowed the metal ion to act as an agonist and activate a GPCR. The coordination of the metal ion binding sites is well characterized in numerous x-ray structures of soluble proteins, and the distances between the chelating atoms and the metal ion are known, providing excellent specific information regarding the orientation of the relative helices (3,8).…”