Chimeric Melatonin mt1 and Melatonin-related Receptors

Abstract

Melatonin receptors bind and become activated by melatonin. The melatonin-related receptor, despite sharing considerable amino acid sequence identity with melatonin receptors, does not bind melatonin and is currently an orphan G protein-coupled receptor. To investigate the structure and function of both receptors, we engineered a series of 14 chimeric receptor constructs, allowing us to determine the relative contribution of each transmembrane domain to ligand binding and receptor function. Results identified that when sequences encoding transmembrane domains 1, 2, 3, 5, or 7 of the melatonin mt 1 receptor were replaced by the corresponding domains of the melatonin-related receptor, the resultant chimeric receptors all displayed specific 2-[ 125 I]iodomelatonin binding. Replacement of sequences incorporating transmembrane domains 4 or 6, however, resulted in chimeric receptors that displayed no detectable 2-[ 125 I]iodomelatonin binding. The subsequent testing of a "reverse" chimeric receptor in which sequences encoding transmembrane domains 4 and 6 of the melatonin-related receptor were replaced by the corresponding melatonin mt 1 receptor sequences identified specific 2-[ 125 I]iodomelatonin binding and melatoninmediated modulation of cyclic AMP levels. To further investigate these findings, site-directed mutagenesis was performed on residues within transmembrane domain 6 of the melatonin mt 1 receptor. This identified Gly 258 (Gly 6.55 ) as a critical residue required for high affinity ligand binding and receptor function.Melatonin is the native ligand for high affinity G proteincoupled melatonin receptors (1, 2). There are now almost 20 reported melatonin receptor sequences, which have been classified into three molecular subtypes, mt 1 , MT 2 , and Mel1c (3). Despite classification as molecular subtypes, they all have essentially identical binding affinities for melatonin (K i ϭ ϳ0.5 nM), and each mediates inhibition of stimulated cyclic AMP levels, mainly but not exclusively via the activation of G i proteins (4). During the cloning of the melatonin receptors a homologue, the melatonin-related receptor, was identified, but despite containing 57% amino acid sequence identity with the TM 1 domains of the melatonin mt 1 receptor, it did not bind 2-[ 125 I]iodomelatonin or melatonin (5, 6). The melatonin-related receptor remains an orphan GPCR with no ligand or signal transduction pathway yet identified.The molecular structure and function of the melatonin mt 1 receptor has been investigated by site-directed mutagenesis, identifying the conserved His residue within TM5 as interacting with the 5-methoxy group of melatonin (7,8). Using the nomenclature of Ballesteros and Weinstein (9), this residue is located at position 5.46 (His 5.46 ), which corresponds to a position identified in the binding site of many other rhodopsin-like GPCRs, including Ser 5.46 (amino acid 207) in the hamster ␤ 2 adrenergic receptor (10). Ligand binding to the ␤ 2 adrenergic receptor has also been shown to involve an ionic interaction ...

GPR50 is the mammalian ortholog of Mel1c: Evidence of rapid evolution in mammals

“…Mutagenesis studies performed either on the human [14-17] or ovine MT1 receptor [18] and studies using human chimeric GPR50/MT1 receptor constructs [19,20] have shown several highly conserved residues in transmembrane helices that are critical for ligand binding especially those in transmembrane helix III (TMIII: S110, G258) (Fig. 3).…”

Homology Models of Melatonin Receptors: Challenges and Recent Advances

“…The importance of residue 5.43 for ligand binding and receptor stabilization is also sustained by site-directed mutagenesis experiments performed on a number of rhodopsin-like receptors [41,42]. Mutation of Gly258 6.55 to threonine brought to a severe loss of 2-iodomelatonin binding affinity and to a remarkable impairment of signal transduction [43,44]. In most GPCR structures, residue 6.55 is located at the C -terminus of TM6, near the extracellular edge of the helical bundle.…”

“…Indeed, Val261 6.58 and Pro265 6.62 formed hydrophobic interactions with the alkyl portion of the propionamide group and the amide oxygen and hydrogen atoms interacted with Tyr175 and Ser182, respectively, located on ECL2. Although no mutagenesis data are available for positions 6.58 and 6.62, the importance of the ECL2 portion in ligand binding to the MT 1 receptor has been assessed in studying chimeric melatonin receptors [44]. Ramelteon did not interact with the two serine residues located on TM3, Ser110 3.35 and Ser114 3.39 , which were shown to be fundamental for ligand stabilization.…”

“…Gly258 6.55 did not directly interact with the ligand, but was located at the entrance of the binding cavity, where it could influence the access of melatonergic ligands. Indeed, site-directed mutagenesis experiments showed that the substitution of Gly258 6.55 with the bulkier threonine brought to a remarkable decrease in ligand binding [43,44]. To evaluate the ability of the MT 1 model to discriminate between agonists and antagonists, different melatonergic ligands were docked within the putative binding region.…”

Towards the Development of Mixed MT1‐Agonist/MT2‐Antagonist Melatonin Receptor Ligands

“…For example, it seems that MT 1 selectivity is favored by replacement of the 5-methoxy group with a larger substituent. [31,41,42] Both site-directed mutagenesis experiments [43][44][45][46] and work with chimeric receptors [47,48] have been employed to investigate the structure-function relationships for MT 1 and MT 2 receptors. Unfortunately, no clear information has been obtained about which amino acids are essential for ligand binding, but there is evidence that agonist and antagonist binding sites are not identical.…”