Mammalian bombesin-like neuropeptides, gastrin releasing peptide (GRP) and neuromedin B (NMB), mediate a diverse range of biological responses in normal tissues, and stimulate the growth of some lung, pancreatic, and prostate carcinomas. Ourlaboratory cloned and characterized three structurally and pharmacologically distinct human bombesin receptors: the gastrin-releasing peptide receptor (GRP-R, or bb2); the neuromedin B receptor (NMB-R, or bb1); and bombesin receptor subtype 3 (BRS-3, or bb3). All three receptors are members of the G-protein coupled receptor superfamily, coupling to heterotrimeric G-proteins that activate phospholipase C. Several advances in our understanding of the function and regulation of these receptors has occurred within the last year: (1) we have used gene targeting strategies to create mice which lack either GRP-R or BRS-3. Mice lacking GRP-R do not exhibit bombesin-induced satiety, implicating the GRP-R as a regulator of appetite. As mice lacking GRP-R age, GRP-R targeted mice are more likely to become obese. In contrast, mice lacking BRS-3 become obese within the first few months of life.We are currently examining the hypothesis that allelic variation in the human BRS-3 gene may contribute to obesity in humans, particulary male individuals. (2) We have developed a protocol that allows us to generate uncoupled receptors embedded in a normal biological membrane. This preparation allows us to reconstitute coupling to purifiedG-proteins, examining the selectivity and enzymology of receptor-catalyzed nucleotide exchange on heterotrimeric GTP-binding proteins. Using this assay, we show that phosphorylation of serines and threonines in the C-terminal domain of the GRP-R inhibits coupling to heterotrimeric G-proteins thereby establishing that ligand-stimulated receptor phosphorylation is the switch that turns off receptor signalling. (3) We have used the yeast two-hybrid system to search for molecules that interact with the C-terminal domain, and have identified several candidates for further study, including one that has been recently shown to interact with the corresponding C-terminal domain of rhodopsin. (4) Using site-directed mutagenesis, we have shown that there are residues in the sixth transmembrane domain that are critical for selective agonist and antagonist binding, and other residues in this same domain that regulate conformational change of the GRP-R. Taken together, these studies implicate the sixth transmembrane domain as an integrator and coordinator of GRP-R function.