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Twodistind receptorsubtypesfor mammalianbombesin-like peptides James Battey and Etsuko Wada JamesBatteyand Etsuko Wadaare at the Laboratoryof Neuroehemistry, NationalInstitute of NeurologicalDiseases and Stroke, Building 36 Rm 4D20, NIH, Bethesda,MD 20892, USA.

524

The mammalian bombesin-like peptides, gastrin- ranatensin, and phyllolitorin subfamilies - based on releasing peptide (GRP) and neuromedin B (NMB), the amino acid sequences of their amidated carboxyare structurally related neurapeptides that elicit a wide terminal octapeptide domains z3, which are the respectrum of biological activities including regulation of &,ions of the peptides critical for receptor binding and smooth muscle contraction, stimulation of secretion, biological activity4-7. At present, two mammalian modulation of neural activity, and growth regulation. bombesin-like peptides, gastrin-releasing peptide Earlier studies have shown that GRP and NMB (GRP) and neuromedin B (NMB), have been identare expressed in different regions of both the CNS ified and characterized. GRP, like the amphibian and peripheral organs. Recent ligand-binding and peptide bombesin, has a leucine residue as its molecular-cloning studies have revealed two pharmaco- penultimate residue, while NMB, like the amphibian logically distinct G-protein-coupled receptor subtypes for peptide ranatensin, has phenylalanine as the penultimammalian bombesin-like peptides that have different mate residue (Fig. 1). No mammalian member of the relative affinities for GRP, NMB and bombesin phyllolitorin subfamily has yet been identified or receptor antagonists. Similar to the peptide ligands, the characterized. two receptor subtypes are expressed in a distinct but Bombesin-like peptides elicit a wide spectrum of overlapping set of CNS regions, some o/which have biological and pharmacological activities, including been identified in functional studies as sites where regulation of smooth muscle contraction and secretion bombesin peptides elicit defined biological responses. of other neuropeptides and hormones. They also Delineation of these peptide ligands and receptor function as mitogens for Swiss 3T3 murine embryonal subtypes will be important in future studies that explore fibroblastss, and have been implicated in the pathothe molecular basis for the heterogeneous nature of the genesis of some human lung carcinomas 9. In the CNS, responses to bombesin observed in mammalian systems. functional studies have indicated that bombesin-like peptides may be important in regulating gastric acid Bombesin is a peptide of 14 amino acids originally secretion, gastrointestinal motility, body temperaisolated from the skin of the European frog Bombma ture, glucose levels, satiety, and certain stereotyped bombina 1. Subsequently, a number of peptides struc- types of behavior such as grooming w-12. More turally and functionally related to bombesin have been recently, GRP, along with other neuropeptides, has purified from amphibians. These peptides have been been implicated in the regulation and maintenance of classified into three subfamilies - the bombesin, circadian rhythms in the suprachiasmatic nucleus 13. At TINS, Vol. 14, No. 12, 1991

BOMBESIN E SUBFAMILY

Bombesin: Rat GRP:

RANATENSIN E SUBFAMILY

Ranatensin: Rat NMB:

o .,

.

""1 T" F SWU L P

S

S

n

LIW Ir Ig_ FlU

Fig. 1. The amino acid sequences of rat gastrin-releasing peptide (GRP) and neuromedin B (NMB) are compared to their

amphibian counterparts, bombesin and ranatensin. Boxes denote carboxy-terminal amino acid residues conserved in these two subfamilies, which are important for both high-affinity receptor binding and biological potency.

(NMB-R) bombesin receptor were isolated, and the primary amino acid sequences determined '.4-~ and compared 26. Structural comparison of the two receptors showed that both receptors encode proteins with predicted molecular masses of about 43 kDa. The two Binding studies s u g g e s t the e x i s t e n c e of at receptors are identical in 56% of their predicted amino least two b o m b e s i n receptor subtypes High-affinity receptors for bombesin peptides have acid sequence; some of the shared residues are also been identified on a variety of cells and tissues, found in a majority of G-protein-coupled receptors including cultured Swiss 3T3 cells 14, rat pituitary activated by ligands other than bombesin peptides ~6. tumor cells 15, pancreatic acinar cells m, esophagus Hydropathy analysis revealed seven stretches of muscularis mucosa 17, gastrin-releasing cells is, and rat hydrophobic amino acids, consistent with a sevenbrain membranes 19. In rat brain, analysis of the membrane-spanning topographic structure conserved relative affinity of bombesin receptors for GRP and in all of the members of the rapidly expanding GNMB has suggested the existence of at least two protein-coupled receptor supeffamily. A comparison pharmacologically distinct populations. The use of of the amino acid sequences of the two receptors, radiolabeled bombesin and NMB in autoradiographic along with the seven putative membrane-spanning analysis of receptors has revealed that bombesin- domains, is shown in Fig. 2. binding sites in some brain regions bind bombesin with higher affinity than NMB, while binding sites in other I regions show higher affinity for NMB than for GR~R 1 MAPNNCSHLNLDVDFFLS..CNDTFNQSLSPPKMDNWFHP~AVY bombesin2°'21. Similar observations were made in the r I J ; I J gastrointestinal tract, where a bombesin receptor on NMB-R l MPPRSLPNLSLPTEASESELEPEVWENDFLPDSDGTTAEL~RC!~'I~S~ esophagus musculafis mucosa bound NMB with 2 higher affinity than either GRP or bombesin~x. By II I II III I ifi contrast, bombesin receptors on pancreatic acinar sl L ~ S ~ L ~ Z Z L T ~ S T M a S W ~ F i S m , ~ G D ~ L ~ T C ' V ~ cells bound GRP and bombesin with higher affinity 3 than NMB 22. Potent and specific bombesin receptor antagonists i01 A S R Y F F D E W v ' F G K L G C ~ ~ T ~ T A ~ R Y R A I V N P M D have provided additional evidence that more than one bombesin receptor subtype must exist. The bombesin 4 receptor antagonist [D-Phe6]bombesin6_~3ethyl ester 149 I Q A S H ~ C ~ I W i ' $ S ~ i ~ E A V F S D L H P F H V K D T N Q T F I S C A I iii i~IIIIll I I I I binds the GRP-preferring bombesin receptor subtype 151 MOTSGV~LWTSLKAVGI~S~AV~EAVFSEV~IGSSD.NSSFTACI (GRP-R) found in pancreas and Swiss 3T3 fibroblasts with high affinity (Ki is in the nanomolar range), and 199 PYPHSNELHPK~S~SEL%~FYVip~SVyyyF~ARNLI QSAYNLPVE efficiently blocks biological responses elicited by this Ill i I ~I~ If If Ill I receptor 23. By contrast, this same antagonist has little effect on responses elicited from the NMB-preferring bombesin receptor (NMB-R) found in esophagus ~2. In 249 GNIHVKKQIESRKRLA~6~.VF~G~C~NH~[~LYRSYHYSEVDTS fact, no antagonist has yet been reported that either I I ;If I III I I I I 25 0 ~E~Tm