EXPRESSION AND CHARACTERIZATION OF CLONED HUMAN BOMBESIN RECEPTORS

Little is known about the pharmacology or cell biology of human bombes in (Bn) receptors, because they are usually present at low levels and both subtypes are frequently present in the same tissues. Human gastri n-releasing peptide (GRP) receptors (huGRP-R) and human neuromedin B ( NMB) receptors (huNMB-R) were stably transfected into BALB/3T3 fibrobl asts. Both receptor types were glycosylated, with 35% of the huGRP-R a nd 38% of the huNMB-R representing carbohydrate residues. The extent o f glycosylation of the transfected huGRP-R was the same as that seen i n the human glioblastoma cell line U-118. Radiolabeled agonist ligands were rapidly internalized, whereas nonintemalized ligand readily diss ociated in a temperature-dependent fashion. The affinities of various agonists for binding to the huGRP-R were Bn (K-i = 1.4 +/- 0.2 nM) = 4 x GRP = 300 x NMB. In contrast, affinities for the huNMB-R were NMB ( K-i = 8.1 +/- 5.2 nM) = 4 x Bn = 600 x GRP. [F-5-D-Phe(6),D-Ala(11)]Bn (6-13)methyl eater was the most potent huGRP-R antagonist, whereas D-N al-Cys-Tyr-D-Trp-Lys-Val-Cys-Nal-NH2 was the most potent huNMB-R antag onist. Agonist binding to either receptor type caused activation of ph ospholipase C and increased cellular [H-3]inositol phosphate levels. G RP was potent at increasing [H-3]inositol phosphate generation in cell s expressing the huGRP-R (EC(50) = 13.6 +/- 1.3 nM), whereas NMB was s imilarly potent when acting upon cells expressing the huNMB-R (EC(50) = 9.3 +/- 1.4 nM). However, neither receptor type, when stimulated wit h agonist, caused an increase in cAMP levels. These data show that sta bly transfected huGRP-R exhibit similar pharmacology for agonists and antagonists, are appropriately glycosylated, and function similarly wi th respect to their ability to alter biological activity, compared wit h natively expressed receptors. Minimal native huNMB-R data are availa ble for comparison, but in general the huNMB-R is similar to the rat N MB receptor in its pharmacology and cell biology.