The principal inhibitory neurotransmitter GABA (gamma-aminobutyric acid) exerts its effects through two ligand-gated channels, GABA(A) and GABA(C) receptors, and a third receptor, GABA(B) , which acts through G proteins to regulate potassium and calcium channels. Cells heterologously expressing the cloned DNA encoding the GABA(B)R1 protein exhibit high-affinity antagonist-binding sites, but they produce little of the functional activity expected from studies of endogenous GABA(B) receptors in the brain. Here we describe a new member of the GABA(B) polypeptide family, GABA(B)R2, that shows sequence homology to GABA(B)R1. Neither GABA(B)R1 nor GABA(B)R2, when expressed individually, activates GIRK-type potassium channels; however, the combination of GABA(B)R1 and GABA(B)R2 confers robust stimulation of channel activity. Both genes are co-expressed in individual neurons, and both proteins co-localize in transfected cells. Moreover, immunoprecipitation experiments indicate that the two polypeptides associate with each other, probably as heterodimers. Several G-protein-coupled receptors (GPCRs) exist as high-molecular-weight species, consistent with the formation of dimers by these receptors, but the relevance of these species for the functioning of GPCRs has not been established. We have now shown that co-expression of two GPCR structures, GABA(B)R1 and GABA(B)R2, belonging to the same subfamily is essential for signal transduction by GABA(B) receptors.

The histamine H 4 receptor is a novel G-protein-coupled receptor with a unique pharmacological profile. The distribution of H 4 mRNA suggests that it may play a role in the regulation of immune function, particularly with respect to allergy and asthma. To define the histamine-binding site of this receptor, molecular modeling and site-directed mutagenesis were used to predict and alter amino acids residing in the histaminebinding pocket. The effects of these alterations on histamine binding and receptor activation were then assessed. (6.52) in TM6 play a role in receptor activation but are not involved in histamine binding. Taken together, these data indicate that although histamine seems to bind to the H 4 receptor in a fashion similar to that predicted for the other histamine receptor subtypes, there are also important differences that can probably be exploited for the discovery of novel H 4 -selective compounds.

Neuromedin U (NmU) is a 25 amino acid peptide prominently expressed in the upper gastrointestinal (GI) tract and central nervous system. It is highly conserved throughout evolution and induces smooth muscle contraction in a variety of species. Our understanding of NmU biology has been limited because the identity of its receptor was unknown. Here we demonstrate that GPR66/FM-3 is specifically stimulated by NmU, causing the mobilization of intracellular calcium. This response was dose-dependent (EC(50) = 10 nM) and specific in that none of over 1000 ligands tested, including other neuromedins (NmB, C, L, K, N), induced a calcium flux in GPR66/FM-3-transfected cells. The GPR66/FM-3 mRNA is prominently expressed in the upper GI tract of humans, as is the mRNA for NmU, consistent with role for this receptor-ligand pair in regulating the function of this organ system. In addition, we show that whereas neuromedin U is expressed by monocytes and dendritic cells, GPR66/FM-3 is expressed by T cells and NK cells. These data suggest a previously unrecognized role for NmU as an immunoregulatory molecule.

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