The end of a myth: cloning and characterization of the ovine melatonin MT(2) receptor.

BACKGROUND AND PURPOSE: For many years, it was suspected that sheep expressed only one melatonin receptor (closely resembling MT(1) from other mammal species). Here we report the cloning of another melatonin receptor, MT(2), from sheep. EXPERIMENTAL APPROACH: Using a thermo-resistant reverse transcriptase and polymerase chain reaction primer set homologous to the bovine MT(2) mRNA sequence, we have cloned and characterized MT(2) receptors from sheep retina. KEY RESULTS: The ovine MT(2) receptor presents 96%, 72% and 67% identity with cattle, human and rat respectively. This MT(2) receptor stably expressed in CHO-K1 cells showed high-affinity 2[(125)I]-iodomelatonin binding (K(D)= 0.04 nM). The rank order of inhibition of 2[(125)I]-iodomelatonin binding by melatonin, 4-phenyl-2-propionamidotetralin and luzindole was similar to that exhibited by MT(2) receptors of other species (melatonin > 4-phenyl-2-propionamidotetralin > luzindole). However, its pharmacological profile was closer to that of rat, rather than human MT(2) receptors. Functionally, the ovine MT(2) receptors were coupled to G(i) proteins leading to inhibition of adenylyl cyclase, as the other melatonin receptors. In sheep brain, MT(2) mRNA was expressed in pars tuberalis, choroid plexus and retina, and moderately in mammillary bodies. Real-time polymerase chain reaction showed that in sheep pars tuberalis, premammillary hypothalamus and mammillary bodies, the temporal pattern of expression of MT(1) and MT(2) mRNA was not parallel in the three tissues. CONCLUSION AND IMPLICATIONS: Co-expression of MT(1) and MT(2) receptors in all analysed sheep brain tissues suggests that MT(2) receptors may participate in melatonin regulation of seasonal anovulatory activity in ewes by modulating MT(1) receptor action.

Cloning and molecular characterization of the novel human melanin-concentrating hormone receptor MCH2.

Using a genomics-based approach for screening orphan G-protein-coupled receptors, we have identified and cloned a novel high-affinity, melanin-concentrating hormone (MCH) receptor. This receptor, named S643b, displays the greatest overall identity (32%) with the previously reported human SLC-1 receptor (MCH1) and to a lesser extent with the somatostatin receptor subtypes. The gene encoding the S643b receptor spans more than 23 kilobase pairs (kb) and was mapped, by radiation hybrid experiments, on chromosome 6q14.3-q15. Comparison of the S643b cDNA with human genomic sequence reveals that the 340-amino-acid receptor is encoded by five exons. Its tissue distribution, as determined by Northern blot and reverse transcription-polymerase chain reaction analysis, indicates that a 4-kb transcript is predominantly expressed in the brain. When expressed in Chinese hamster ovary (CHO) cells, the S643b receptor displays a strong, dose-dependent, transient elevation of intracellular calcium in response to MCH (EC(50) = 9.5 nM). During the present study, we isolated a splice variant, designated S643a, encoding for a receptor that was not activated by MCH in a cellular calcium mobilization assay. Comparative pharmacological studies using CHO cells stably expressing either SLC-1 or S643b receptors demonstrated that similar structural features of MCH are required to stimulate intracellular Ca(2+) mobilization at both receptors. The identification and localization of this new MCH receptor (MCH2) provides further insight into the physiological implication of MCH in modulating behavioral responses, including food intake.

Genomic organization and characterization of splice variants of the human histamine H3 receptor.

In the present paper we report the genomic organization of the human histamine H3-receptor gene, which consists of four exons spanning 5.5 kb on chromosome 20. Using PCR, six alternative splice variants of the H3 receptor were cloned from human thalamus. These variants were found to be coexpressed in human brain, but their relative distribution varied in a region-specific manner. These isoforms displayed either a deletion in the putative second transmembrane domain (TM), H3(DeltaTM2, 431aa) or a variable deletion in the third intracellular loop (i3), H3(Deltai3, 415aa), H3(Deltai3, 365aa), H3(Deltai3, 329aa) and H3(DeltaTM5+Deltai3, 326aa). In order to determine the biological role of the H3 receptor variants compared with the 'original' H3(445aa) receptor, three isoforms, namely H3(445aa), H3(DeltaTM2, 431aa) and H3(Deltai3, 365aa), were expressed in CHO cells and their pharmacological properties were investigated. Binding studies showed that H3(DeltaTM2, 431aa) transiently expressed in CHO cells was unable to bind [125I]iodoproxyfan, whereas both the H3(445aa) and H3(Deltai3, 365aa) receptors displayed a high affinity for [125I]iodoproxyfan [K(d)=28+/-5 pM (n=4) and 8+/-1 pM (n=5) respectively]. In addition, H3(Deltai3, 365aa) possessed the same pharmacological profile as the H3(445aa) receptor. However, in CHO cells expressing H3(Deltai3, 365aa), H3 agonists did not inhibit forskolin-induced cAMP production, stimulate [35S]guanosine 5'-[gamma-thio]triphosphate ([35S]GTP[S]) binding or stimulate intracellular Ca(2+) mobilization. Therefore the 80-amino-acid sequence located at the C-terminal portion of i3 plays an essential role in H3 agonist-mediated signal transduction. The existence of multiple H3 isoforms with different signal transduction capabilities suggests that H3-mediated biological functions might be tightly regulated through alternative splicing mechanisms.

Structure-activity relationship studies of melanin-concentrating hormone (MCH)-related peptide ligands at SLC-1, the human MCH receptor.

Melanin-concentrating hormone (MCH) is a cyclic nonadecapeptide involved in the regulation of feeding behavior, which acts through a G protein-coupled receptor (SLC-1) inhibiting adenylcyclase activity. In this study, 57 analogues of MCH were investigated on the recently cloned human MCH receptor stably expressed in HEK293 cells, on both the inhibition of forskolin-stimulated cAMP production and guanosine-5'-O-(3-[(35)S]thiotriphosphate ([(35)S]- GTPgammaS) binding. The dodecapeptide MCH-(6-17) (MCH ring between Cys(7) and Cys(16), with a single extra amino acid at the N terminus (Arg(6)) and at the C terminus (Trp(17))) was found to be the minimal sequence required for a full and potent agonistic response on cAMP formation and [(35)S]- GTPgammaS binding. We Ala-scanned this dodecapeptide and found that only 3 of 8 amino acids of the ring, namely Met(8), Arg(11), and Tyr(13), were essential to elicit full and potent responses in both tests. Deletions inside the ring led either to inactivity or to poor antagonists with potencies in the micromolar range. Cys(7) and Cys(16) were substituted by Asp and Lys or one of their analogues, in an attempt to replace the disulfide bridge by an amide bond. However, those modifications were deleterious for agonistic activity. In [(35)S]- GTPgammaS binding, these compounds behaved as weak antagonists (K(B) 1-4 microm). Finally, substitution in MCH-(6-17) of 6 out of 12 amino acids by non-natural residues and concomitant replacement of the disulfide bond by an amide bond led to three compounds with potent antagonistic properties (K(B) = 0.1-0.2 microm). Exploitation of these structure-activity relationships should open the way to the design of short and stable MCH peptide antagonists.

Truncated isoforms inhibit [3H]prazosin binding and cellular trafficking of native human alpha1A-adrenoceptors.

We have identified from human liver eight alpha(1A)-adrenoceptor (alpha(1A)-AR) splice variants that were also expressed in human heart, prostate and hippocampus. Three of these alpha(1A)-AR isoforms (alpha(1A-1)-AR, alpha(1A-2a)-AR and alpha(1A-3a)-AR) gave rise to receptors with seven transmembrane domains (7TMalpha(1A)-AR). The other five (alpha(1A-2b)-AR, alpha(1A-2c)-AR, alpha(1A-3c)-AR, alpha(1A-5)-AR and alpha(1A-6)-AR) led to truncated receptors lacking transmembrane domain VII (6TMalpha(1A)-AR). The 7TMalpha(1A)-AR isoforms transiently expressed in COS-7 cells bound [(3)H]prazosin with high affinity (K(d) 0.2 nM) and mediated a noradrenaline (norepinephrine)-induced increase in cytoplasmic free Ca(2+) concentration, whereas the 6TMalpha(1A)-AR isoforms were incapable of ligand binding and signal transduction. Immunocytochemical studies with N-terminal epitope-tagged alpha(1A)-AR isoforms showed that the 7TMalpha(1A)-AR isoforms were present both at the cell surface and in intracellular compartments, whereas the 6TMalpha(1A)-AR isoforms were exclusively localized within the cell. Interestingly, in co-transfected cells, each truncated alpha(1A)-AR isoform inhibited [(3)H]prazosin binding and cell-surface trafficking of the co-expressed 'original' 7TMalpha(1A-1)-AR. However, there was no modification of either the [(3)H]prazosin-binding affinity or the pharmacological properties of alpha(1A-1)-AR. Immunoblotting experiments revealed that co-expression of the alpha(1A-1)-AR with 6TMalpha(1A)-AR isoforms did not impair alpha(1A-1)-AR expression. Therefore the expression in human tissues of many truncated isoforms constitutes a new regulation pathway of biological properties of alpha(1A)-AR.