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We report the discovery, tissue distribution and pharmacological characterization of a novel receptor, which we have named H4. Like the three histamine receptors reported previously (H1, H2, and H3), the H4 receptor is a G protein-coupled receptor and is most closely related to the H3 receptor, sharing 58% identity in the transmembrane regions. The gene encoding the H4 receptor was discovered initially in a search of the GenBank databases as sequence fragments retrieved in a partially sequenced human genomic contig mapped to chromosome 18. These sequences were used to retrieve a partial cDNA clone and, in combination with genomic fragments, were used to determine the full-length open reading frame of 390 amino acids. Northern analysis revealed a 3.0-kb transcript in rat testis and intestine. Radioligand binding studies indicated that the H4 receptor has a unique pharmacology and binds [(3)H]histamine (K(d) = 44 nM) and [(3)H]pyrilamine (K(d) = 32 nM) and several psychoactive compounds (amitriptyline, chlorpromazine, cyproheptadine, mianserin) with moderate affinity (K(i) range of 33-750 nM). Additionally, histamine induced a rapid internalization of HA-tagged H4 receptors in transfected human embryonic kidney 293 cells.

Numerous mutant G protein-coupled receptors with diminished or no function have been described that are naturally occurring or that are the product of gene manipulation. It has largely been assumed that receptor mutants do not affect the function of the wild-type receptor; however, the occurrence of G protein-coupled receptor dimerization suggests the possibility that an intermolecular interaction between mutant and wild-type receptors can occur. We have shown previously that the D2 dopamine receptor (D2DR) exists as dimers in cell lines and brain tissue. In this study, we demonstrated that mutant D2DR can modulate the function of the wild-type D2DR. While attempting to elucidate the structure of the D2DR dimer, we demonstrated that nonfunctional D2DR substitution and truncation mutants antagonized wild-type D2DR function. Furthermore, from analyses of this interaction between the receptor mutants and the D2DR, using photoaffinity labeling, we provide evidence that the D2DR is oligomeric in the cell.

Two G protein-coupled receptors (Edg-2) and (Edg-4) for the lysolipid phosphoric acid mediator lysophosphatidic acid have been described by molecular cloning. However, the calcium-mobilizing receptor Edg-4 is not expressed in some cell lines that exhibit robust calcium responses to this ligand, thus predicting the existence of additional receptor subtypes. We report here on the characterization of a third human lysophosphatidic acid receptor subtype, Edg-7, which mediates lysophosphatidic acid-evoked calcium mobilization. In a rat hepatoma Rh7777 cell line that lacks endogenous responses to lysophosphatidic acid, this lipid mediator, but not others, evokes calcium transients when the cells have been transfected with Edg-7 or Edg-4 DNAs. Furthermore, frog oocytes exhibit a calcium-mediated chloride conductance in response to mammalian-selective lysophosphatidic acid mimetics after injection of Edg-7 mRNA. Edg-7-expressing Rh7777 cells do not show inhibition of forskolin-driven rises in cAMP in response to lysophosphatidic acid. However, membranes from HEK293T cells cotransfected with Edg-7 and G(i2)alpha protein DNAs show lysophosphatidic acid dose-dependent increases in [gamma-(35)S]GTP binding with an EC(50) value of 195 nM. When we used this assay to compare various synthetic LPA analogs at Edg-2, Edg-4, and Edg-7 receptors, we found that ethanolamine-based compounds, which are full LPA mimetics at Edg-2 and Edg-4, exhibit little activity at the Edg-7 receptor. Edg-7 RNA was detected in extracts of several rat and human tissues including prostate. Together, our data indicate that Edg-7 is a third lysophosphatidic acid receptor that couples predominantly to G(q/11)alpha proteins.

An analysis of configurational effects on the 13C chemical shifts of bicyclic compounds of the 1,6-anhydrohexopyranose and 2,7-anhydroheptulopyranose type is presented. As with other saturated ring Systems, strong upfield shifts are found in association with an axial orientation of the substituents, although in this respect 13C nuclei of the anhydro ring appear to exhibit atypical behaviour. Deshielding changes characterize a syn-diaxial orientation of hydroxyl groups, and also of a hydroxyl group with respect to the bridgehead bonds. No appreciable effects of antiperiplanarhydroxyl groups on 13C chemical shifts have been detected. Possible interrelationships between 13C or 1H chemical shifts and steric interactions are discussed with reference to the relative stability of isomeric anhydrosugars and C—H bond polarization. Some characteristics of one-, two-, and three-bond 13C–1H coupling are described, and are shown to be of value in several instances for the assignment of 13C signals. The 1H-coupled signal for methylenic C-6 of 1,6-anhydrides is commonly non-first order, in contrast to the simpler pattern exhibited by methylenic C-1 of 2,7-anhydrides.