Takao Tashiro

The recent cloning of three melanocortin receptors suggests an unexpected diversity in this family of seven transmembrane G-protein linked receptors. Herein, we report the cloning, expression, and gene localization of a fourth human melanocortin receptor, the melanocortin-4 receptor. By Northern blot analysis and in situ hybridization, this receptor is expressed primarily in the brain, but its expression is notably absent in the adrenal cortex, melanocytes, and placenta. Agonist stimulation of COS-1 cells transiently transfected and L-cells permanently transfected with the coding region of the cloned melanocortin-4 receptor leads to increases in intracellular cyclic 3',5'-adenosine monophosphate. The profile of the responses of the melanocortin-4 receptor to different melanocortins distinguishes it from melanocortin receptors previously described. Using the technique of fluorescent in situ hybridization, the gene encoding the melanocortin-4 receptor was localized to chromosome 18 (q21.3).

Using the technique of the polymerase chain reaction primed with oligonucleotides based on the homologous transmembrane regions of seven transmembrane G protein-linked receptors, we isolated three full-length human genes that encode a novel subgroup of this receptor family. Recently, two of these receptors were identified as specific for alpha-melanocyte-stimulating hormone (alpha-MSH) and adrenocorticotropic hormone. We report the molecular cloning and pharmacologic characterization of a third member of this subgroup. The gene for this receptor encodes a protein of 361 amino acids in length. Its pharmacology characterizes it as an MSH receptor specific to the heptapeptide core common to adrenocorticotropic hormone and alpha-, beta-, and gamma-MSH. By Northern blot hybridization and polymerase chain reaction, it is expressed in brain, placental, and gut tissues but not in melanoma cells or in the adrenal gland. These findings may yield insight into the physiology of peptides derived from pro-opiomelanocortin post-translational processing.

We undertook these studies to characterize the molecular basis of the interaction of histamine with the H2 receptor. Key areas of homology in the structures of the histamine H2 and beta 2 adrenergic receptor suggested specific transmembrane amino acids that might be important for binding of histamine. A third transmembrane aspartic acid of the histamine receptor (Asp98), thought to serve as a counter anion that interacts with the cationic amine moiety of histamine, was mutated to Asn98, and the mutated receptor was expressed in Hepa cells. Removal of the negatively charged amino acid abolished both binding of the H2 receptor antagonist [methyl-3H]tiotidine and histamine stimulated increases in cellular cAMP content. Mutation of a fifth transmembrane aspartic acid (Asp186) to Ala186 or Asn186 by itself or in conjunction with mutation of another fifth transmembrane amino acid (Thr190 to Ala190) resulted in a loss of [methyl-3H] tiotidine binding, although the generation of cAMP in response to histamine was maintained. The histamine receptor with only a Thr190 to Ala190 or Cys190 mutation retained the ability to bind [methyl-3H]tiotidine, but both the affinity and efficacy of binding were reduced. These data lead us to propose a model for histamine binding in which Asp98 is essential for histamine binding and action, Asp186 defines H2 selectivity, and Thr190 is important in establishing the kinetics of histamine binding, but is not essential for H2 selectivity.

Serum pepsinogen levels were measured in 137 stomach cancer patients and compared with those of 288 normal cancer-free subjects. The serum pepsinogen levels of stomach cancer patients, especially pepsinogen I and the pepsinogen I/pepsinogen II ratio were significantly lower than those of normal controls and correlated well with the extent of chronic gastritis associated with the cancerous stomach. These results were in good accordance with the results of previous studies indicating that the cancer derived from the stomach where chronic gastritis/intestinal metaplasia is extensive. The high sensitivity and specificity of this non-invasive serum test to detect chronic gastritis suggested the possibility of its application to the mass screening of stomach cancer.