Expression cloning of a high-affinity melatonin receptor from Xenopus dermal melanophores.

Abstract

Using an expression cloning strategy, a highanit meltonin receptor cDNA has been isolated from Xenopus laei dermal melanophores. Transient expression of the cDNA in COS-7 cells resulted in h-affinity 2-[125I-odomneatonn binding (Kd = 6.3 ± 0.3 x 10-11 M). In addition, six ligands exhibited a rank order of inhibition of specific 2-uI has revealed the presence of high-affinity melatonin receptors (Kd < 2 x 10-10 M) (4-6). Receptor affinity is sensitive to guanine nucleotides and activation of the receptors consistently leads to inhibition of adenylyl cyclase through a pertussis toxin-sensitive mechanism (7-11). High-affinity melatonin receptors thus appear to belong to the superfamily of guanine nucleotide binding protein (G protein)-coupled receptors. To better understand how melatonin acts at a cellular and molecular level, it is important to determine melatonin receptor structure.One of the earliest described actions of melatonin is its ability to cause melanin aggregation in dermal melanophores of amphibians (for review, see ref. 12). This action is mediated through a high-affinity melatonin receptor that is coupled to inhibitory G protein (Gi) (13,14). Using a cDNA library constructed from an immortalized cell line ofXenopus dermal melanophores and a mammalian cell expression cloning strategy, we have succeeded in cloning a high-affinity melatonin receptor. The cDNA encodes a protein that is a newly discovered member of the G protein-coupled receptor family. 11 MATERIALS AND METHODS Expression Cloning. Xenopus laevis dermal melanophores were cultured as described (15). The melanophores were derived from a clonal line that was isolated from a primary culture. Total cellular RNA was isolated from melanophores by extraction with guanidinium thiocyanate and separation in cesium chloride. Melanosomes were removed as described before centrifugation on the cesium chloride gradient (16). Poly(A)+ RNA was isolated by established methods (17).A random-primed cDNA library was constructed with a kit from Pharmacia. Double-stranded cDNA was ligated with nonpalindromic BstXI/EcoRI adaptors (Invitrogen). The cDNA was size-fractionated on an agarose gel, and cDNA>2 2 kb was recovered by electroelution. The size-selected cDNA was ligated into the expression vector pcDNAI (Invitrogen) and introduced into Escherichia coli MC1061/P3 by electroporation.A total of 4 x 105 recombinants were obtained from 5 pg of poly(A)+ RNA and divided into 54 pools of "7400 clones each. Plasmid DNA was prepared from each pool by the alkaline lysis method and transfected into COS-7 cells by the DEAE-dextran method (18). Three days after transfection, cells were incubated with 90 pM 125I-Mel in 50 mM Tris-HCl (pH 7.4) containing 100 mM NaCI, 5 mM KCl, 2 mM CaCl2, and 5% Nu-Serum I (Collaborative Biomedical Products, Bedford, MA) for 2 hr at room temperature. Cells were washed, air dried, and exposed to x-ray film for 14 days. A pool of clones that showed positive signals was subdivided, and the transfection procedure was repeated. This ...

Stimulation of cAMP accumulation by the cloned Xenopus melatonin receptor through Gi and Gz proteins

“…1A), indicating the involvement of Gi proteins. Our results are in accordance with similar reports using NIH-3T3 or CHO cells [2,3].…”

“…The recent cloning of the melatonin receptor from Xenopus [2] and rat [3] has confirmed the long-suspected notion that this receptor belongs to the superfamily of G protein-coupled receptors. Numerous reports suggest that melatonin can inhibit adenylyl cyclase via pertussis toxin (PTX) sensitive Gi-like proteins [4,5].…”

“…The cloned Xenopus melatonin receptor has been shown to inhibit adenylyl cyclase in transfected CHO cells [2]. We first begun by ensuring that the Xenopus melatonin receptor behaves similarly in transfected human embryonic kidney 293 cells.…”

“…The cDNA encoding Xenopus melatonin receptor [2], a generous gift from Dr. Steve Reppert (Massachusetts General Hospital, Boston), was subcloned into pcDNAI (Invitrogen) at EeoRI sites, cDNAs encoding the ~ subunit of G~2 and Gt3 in pCIS were kindly provided by Dr. Melvin Simon (California Institute of Technology, Pasadena). The cDNA encoding AC II in pcDNAI was from Dr. Randall Reed (Johns Hopkins University, MD).…”

“…Numerous reports suggest that melatonin can inhibit adenylyl cyclase via pertussis toxin (PTX) sensitive Gi-like proteins [4,5]. Expression of the cloned melatonin receptors in NIH-3T3 and CHO cells has revealed that this receptor can indeed inhibit adenylyl cyclase in a PTX-sensitive manner [2,3].…”