(Received for publication, September 13, 1995; and in revised form, January 9, 1996)
From the
Amphibian bombesin is the prototypic peptide that defines the
bombesin-like peptide family. In this paper we show that in the frog Bombina orientalis, there are actually 3 distinct forms of
bombesin, and each of these peptides is an agonist with differing
affinities for the known bombesin receptors. Oligonucleotides
complementary to the 5`- and 3`-untranslated regions of the bombesin
mRNA were used to amplify bombesin-related cDNAs from the skin, brain,
and gut of B. orientalis. Three classes of cDNAs were found.
One class encoded the previously characterized form of bombesin which
has a Leu at position 13 ([Leu]bombesin). The
other two classes, respectively, encoded new bombesin-like peptides
which we have designated as [Phe
]bombesin and
[Ser
,Arg
,Phe
]bombesin
([SAP]bombesin). The existence of [SAP]bombesin in
skin was confirmed by tandem mass spectrometry. Polymerase chain
reaction analysis of genomic DNA showed the mRNAs for
[Leu
]bombesin,
[Phe
]bombesin, and [SAP]bombesin most
likely arise from separate genes. Polymerase chain reaction analysis
showed different patterns of tissue-specific expression for each form.
[Leu
]Bombesin and [SAP]bombesin were
predominantly expressed in skin, brain, and gut;
[Phe
]bombesin was expressed only in brain, and
[Leu
]bombesin predominated in oocytes.
[SAP]Bombesin contained a cleavage site between residues 4
and 5, which if used would yield the peptide
[SAP]bombesin(5-14) which has the sequence
[Gln
,Arg
]neuromedin B. Thus a frog
homolog of NMB could derive from the [SAP]bombesin
prohormone. [Phe
]Bombesin,
[SAP]bombesin, and [SAP]bombesin(5-14) were
synthesized and their affinities for the mammalian bombesin-like
peptide (GRP and NMB) receptors determined. These peptides acted as
agonists for the GRP and NMB receptors, with relative potencies for the
GRP receptor of [Leu
]bombesin >
[Phe
]bombesin >
[SAP]bombesin(5-14) > [SAP]bombesin and
for the NMB receptor of [Phe
]bombesin >
[SAP]bombesin(5-14) >
[Leu
]bombesin > [SAP]bombesin. None
of these peptides demonstrated high affinity binding for the BRS-3
receptor. The different receptor affinities and tissue distribution of
these peptides suggests distinct physiologic roles and raises the
possibility of as yet uncharacterized mammalian homologs of these new
amphibian peptides.
Bombesin, a tetradecapeptide, was isolated from the skin of the
frog Bombina bombina by Anastasi et al.(1) in 1971 (Fig. 1). The bombesin-related peptides
ranatensin and [Leu]phyllolitorin were
subsequently isolated from the skin of Rana and Phyllomedusa species by Nakajima et al.(2) and by Yasuhara et al.(3) (Fig. 1). Amphibian bombesin was found to have
multiple effects in mammals (4, 5) and bombesin-like
immunoreactivity was observed in mammalian brain, GI tract, and
lung(6, 7, 8) . In 1979, using gastrin
release as a bioassay McDonald and co-workers (9) isolated from
porcine stomach a 27-amino acid peptide homologous to the carboxyl
terminus of bombesin and named it gastrin-releasing peptide (GRP) (
)(Fig. 1). GRP is widely distributed in mammals;
acts as a neurotransmitter in brain, a paracrine hormone in GI tract,
and a growth factor in developing
lung(10, 11, 12) . Neuromedin B (NMB) is a
second mammalian bombesin-like peptide (Fig. 1). NMB was
originally isolated from porcine spinal cord (13) and like GRP
is widely distributed in brain and GI tract. Frogs have both GRP and
bombesin (14) indicating they are distinct peptides. In frogs
GRP predominates in gut, bombesin in skin and brain(14) .
Phyllogenetic analysis of the prohormones for
GRP(14, 15, 16) ,
NMB(17, 18) , ranatensin (17) ,
bombesin(19) , and phyllolitorin (20) shows the
bombesin-like peptides can be divided into three distinct branches. The
peptides originally found in skin, bombesin, ranatensin, and
phyllolitorin form one branch which we have designated as the
bombesin-related peptide (BRP) branch; GRP and NMB form the other two
branches(20) . To date the BRP family has only been
characterized in amphibians, although given that the three branches
appear to have diverged prior to the vertebrate radiation, mammalian
homologs of the BRP peptides may exist(20) .
Figure 1: The three subfamilies of bombesin-like peptides. Representative members of each subfamily are shown. Bombesin, ranatensin, and phyllolitorin are amphibian, GRP and NMB are from the species shown. GRP-10 is the COOH-terminal decapeptide of GRP and contains full biological activity of GRP.
Four subtypes of receptors for bombesin-like peptides have been characterized to date. A GRP-preferring subtype was cloned by Spindel et al.(21) and by Battey et al.(22) ; a neuromedin B receptor was cloned by Wada et al.(23) ; and a third subtype whose ligand is still unknown, designated BRS-3, was cloned by Fathi et al.(24) and by Gorbulev et al.(25) . Our laboratory recently characterized a fourth class of bombesin receptors (designated BB4) which appear to constitute the receptors for the bombesin-related peptides as they have higher affinity for bombesin than GRP(26) . A second nomenclature system for the bombesin receptors is also in use. In this system, the NMB receptor is known as the BB1 receptor, the GRP receptor is the BB2 receptor, and the BRS-3 receptor is the BB3 receptor(27, 28) .
While bombesin-related peptides
have been described in multiple species of frogs, it has previously
been believed that a given species has one primary BRP, e.g. bombesin in Bombina orientalis,
ranatensin in Rana pipiens, and litorin in Litoria
aurea(29) , and that these peptides will have either a Leu
or a Phe as the penultimate residue. An exception to this rule was Phyllomedusa sauvagei which expresses both
[Leu]phyllolitorin and
[Phe
]phyllolitorin. In cloning the cDNAs encoding
[Leu
]phyllolitorin and
[Phe
]phyllolitorin we observed that the cDNAs
differed by only a single nucleotide. This suggested that other species
of frogs might also express multiple BRP-encoding cDNAs that may
previously have not been detected because of their high homology. To
examine this, reverse transcription-PCR (RT-PCR) was used to amplify
bombesin-related cDNAs in B. orientalis. Sequence
analysis then revealed three different bombesin-related cDNAs, each
encoding different subtypes of bombesin. The bombesin subtypes have
different distributions and receptor affinities which suggests
different physiologic roles for these multiple peptide forms.
Figure 2:
A,
nucleotide and amino acid sequence of the 3 bombesin mRNAs. Nucleotide
sequence derived from sequencing at least 4 independent clones of each
form. Coding region for bombesin, [Phe]bombesin,
and [Leu
]bombesin is boxed. The
proteolytic cleavage sites for the bombesin-like peptides are underlined. The location of the common 5` and 3` primers used
to amplify the 3 cDNAs for the distribution studies are as shown. The
location of the [Phe], [Leu], and
[SAP]bombesin specific primers for differential hybridization
are as shown. B, the sequences of the three forms of bombesin
are as shown. [SAP]Bombesin(5-14) would be produced if
cleavage of [SAP]bombesin occurred at the internal Ser-Leu
motif that is used for cleavage of
bombesin.
In order to
differentiate between related bombesin cDNAs, RNA was reverse
transcribed as above, then amplified with primers common to all cDNAs.
10 µl of the reaction was then electrophoresed in triplicate on 1%
agarose, blotted, and hybridized to 20- or 21-base oligonucleotides
specific for each cDNA. Hybridization conditions were as described by
Sommer et al.(31) using 25% formamide, 5 SSC,
40 °C; washing was in 0.5
SSC, 55 °C. Specificity of
hybridization was checked by including samples of each cDNA on each
gel. Sequence of primers used is shown in Fig. 2.
Individual HPLC fractions (average volume 0.4 ml) were
analyzed by matrix-assisted laser desorption ionization (MALDI) mass
spectrometry (32) , using a Voyager MALDI time-of-flight (TOF)
mass spectrometer (PerSeptive Biosystems, Framingham, MA). For these
experiments, a 1-µl aliquot of each HPLC fraction was mixed with
0.5 µl of a matrix solution, placed on 1 well of a 100-well
stainless steel sample plate and allowed to dry at room temperature.
The matrix solution was a 10 mg/ml solution of
-cyano-4-hydroxycinnamic acid in CH
CN/ethanol (1/1).
Samples were irradiated with a pulsed 337-nm N
laser and
20-50 scans were summed. The instrument was calibrated using
phosphokemptide and oxidized insulin
chain as external standards,
and typical mass measurement accuracy was better than 0.1%.
Fraction
22, which contained the molecular ion of interest (m/z 1643,
average MH), was further analyzed by post source decay
(PSD) on a Voyager RP MALDI-TOF instrument with a reflector (PerSeptive
Biosystems, Framingham, MA). Under PSD conditions (33) , one
observes fragmentation of protonated peptides that are very similar to
those described for high and low energy collision-induced dissociation
(CID)(34) . In addition, this same fraction was analyzed by
liquid secondary ion mass spectrometry under CID conditions to obtain
additional sequence information. To carry out this latter experiment a
small aliquot of the HPLC fraction (20 µl) was dried down onto a
stainless steel probe tip and dissolved in a mixture of
glycerol/thioglycerol (2/1). The samples were then acidified with 0.1
µl of 1% trifluoroacetic acid and inserted into the source of a
Kratos Concept IIHH mass spectrometer as described in detail elsewhere (35) .
RT-PCR of RNA from skin, brain, and stomach of B.
orientalis with primers spanning the coding region of the bombesin
cDNA resulted in bands clearly visible by staining with ethidium
bromide. These bands were subcloned and multiple clones from skin,
brain, and stomach were sequenced. As shown in Fig. 2, three
classes of cDNAs were obtained. These cDNAs were highly homologous, but
the minor nucleotide differences resulted in new forms of bombesin. One
class of cDNAs was the cDNA for [Leu]bombesin as
described previously by our laboratory (19) . The second class
of cDNAs contained a G to C change in the codon at the Leu
position of bombesin, such that the cDNA encoded the new
bombesin-like peptide, [Phe
]bombesin. The third
class contained three nucleotide changes in the bombesin coding region
to produce the new peptide,
[Ser
,Arg
,Phe
(SAP)]bombesin.
The sequences of the three forms of bombesin are shown in Fig. 2B. Interestingly, the Ser
substitution in [SAP]bombesin creates another copy of
the Ser-Leu motif that signals cleavage of bombesin from its precursor.
If this cleavage site is utilized, then the peptide
[Gln
-Arg
]neuromedin B which resembles
mammalian neuromedin B could be produced from the
[SAP]bombesin precursor (Fig. 2B). To
determine the representation of the three cDNA forms in genomic DNA,
the same primers were used to amplify genomic DNA prepared from B. orientalis liver. Sequence analysis of the
resulting product showed roughly equal representation of the three
forms suggesting the existence of multiple genes.
Given that
sequence analysis of multiple clones amplified from both genomic DNA
and reverse-transcribed cDNA showed the identical 3 classes of cDNAs,
the possibility of PCR artifact is very unlikely. To definitively prove
this, advantage of the high levels of peptide present in skin was taken
and mass spectrometry was performed to show the existence of
[SAP]bombesin in skin. HPLC separation of the B. orientalis skin extract was performed as previously
reported(41) . Mass spectrometric profiling of the individual
HPLC fractions allowed for the identification of a fraction eluting at
about 25% CHCN (Fraction 22). The MALDI mass spectrum of
this fraction contained a peak at m/z 1643, that was close to
the expected value for [SAP]bombesin, i.e. MH
at m/z 1642.8 as well
as peaks 16 and 32 Da higher at m/z 1659 and 1675. Subsequent
analysis of this same fraction at a higher resolving power (M/
M
2, 500) using the Voyager RP TOF mass spectrometer resulted in the
mass resolution of the isotopic pattern of this molecular ion and the
assignment of the
C-containing monoisotopic mass at m/z 1641.8, agreeing exactly with the expected value of m/z 1641.8. The two species at slightly higher mass are likely
oxidized versions of this same peptide containing methionine sulfoxide
(+16 Da, O) and sulfone (+32 Da, O
), as their
relative abundance increased during storage (data not shown).
Sequence analysis by PSD and high energy CID of the (non-oxidized)
peptide yielded information supported the identity of this peptide as
the [SAP]bombesin analog. The PSD spectrum shown in Fig. 3contains a number of low mass immonium ions, NH
= CHR (42) and/or
related ions for the expected amino acids Trp (m/z 159 and
130), Arg (m/z 112, 70), His (m/z 110), Gln (m/z 101), and Leu (m/z 86). In addition, a strong b-ion series dominates the rest of the PSD spectrum, resulting
from cleavage at the amide nitrogens with charge retention at the
NH
terminus(43) . These ions, along with some a- and y-ions, defined 13 of the 14 amino acids of the
[SAP]bombesin sequence (see Fig. 3, inset).
The high energy CID spectrum (not shown) added additional support for
the composition, containing distinctive immonium ions for all amino
acids except for Gly. In addition, a partial sequence of
(pGlu,Gln)-Ser-Leu-Gly-Asn-Gln-Trp-(Ala,Arg)-Gly-His-Phe-Met-amide
could be determined based on abundant a, b, and c-type ions. Taken together, the PSD and high energy CID data
provided sufficient sequence data to confirm the identity of this
peptide as that for [SAP]bombesin as predicted from the cDNA.
This also confirms that processing at the atypical Ser-Leu site takes
place as predicted to yield [SAP]bombesin.
Figure 3: Post source decay MALDI-TOF spectrum of the selected m/z 1642.8 (average) ion with sequence ions labeled according to the accepted nomenclature for peptide fragmentation(43) . Additional ions were also observed that are consistent with double cleavage reactions forming a series of internal peptide ions. These latter ions are labeled according to single letter amino acid codes; e.g. GH is Gly-His at m/z 195, where the prime refers to the additional loss of 28 Da (CO).
The relative
distribution of each bombesin subtype was examined by RT-PCR. Common
primers were used to amplify all three bombesin cDNAs, then primers
specific for each form were used for specific hybridization. As shown
in Fig. 4, [Leu]bombesin was present in
most tissues (not heart or liver), with highest levels in skin and
brain. [Leu
]Bombesin was the predominant form of
bombesin present in oocytes. [SAP]Bombesin was also widely
distributed, with highest levels in brain. Interestingly,
[SAP]bombesin was also present in lung.
[Phe
]Bombesin was present only in skin. The
distribution in brain was further examined in dissected brain regions (Fig. 5). [Leu
]Bombesin was present in
all regions examined. [Phe
]Bombesin was also
present throughout brain, although little was present in pituitary.
[SAP]Bombesin had a more limited distribution, with higher
levels in spinal cord and pituitary, and lower levels in other brain
regions. The tissue-specific patterns of distribution suggests
different physiologic functions for each peptide. This is further
supported by the different affinities of the peptides for the known
bombesin receptors.
Figure 4:
RT-PCR
analysis of the expression of the [Leu bombesin (Leu), [SAP]bombesin (SAP), and
[Phe
]bombesin (Phe) mRNAs in the frog
tissues shown. Tissues were reverse transcribed with the 5` and 3`
primers shown in Fig. 2A, then hybridized with the
specific primers shown in Fig. 2A. Controls were
reverse-transcription reactions lacking reverse transcriptase
(-RT) from skin, brain, pancreas (panc), lung,
and oocytes. Controls also included reverse transcription reaction with
no input RNA (No RNA) and PCR amplification with no DNA (No DNA). Positive controls to show specificity were cDNAs
amplified from the [Leu
]bombesin cDNA,
[Phe
]bombesin cDNA, and [SAP]bombesin
cDNA.
Figure 5: RT-PCR analysis of dissected brain regions as shown. Hypo, hypothal = hypothalamus. Primers and controls as for figure 3.
The ability of these new bombesin-like peptides
to act as agonists at the known bombesin receptors was tested in Xenopus oocytes expressing the GRP, NMB, and BRS-3 receptors.
As shown in Fig. 6, the new bombesin-like peptides,
[Phe]bombesin, [SAP]bombesin, and
[SAP]bombesin(5-14) were potent agonists for the GRP
and NMB receptors. [Phe
]Bombesin is a
particularly potent agonist both for the GRP and NMB receptor. None of
these peptides showed agonist activity for the BRS-3 receptor (data not
shown). While expression in oocytes provides an index of agonist
potency, it is not quantitative. Thus K
values to
displace binding of
I-labeled bombesin to GRP and NMB
receptors in AR42J and rat olfactory membranes, respectively, was
measured (Fig. 7, Table 1). GRP receptor binding was
measured by displacement of
I-bombesin (concentration
= 0.05 nM) from membranes prepared from AR42J cells and
NMB receptor binding was measured by displacement of
I-bombesin from membranes prepared from rat olfactory
bulb. Nonspecific binding was consistently less than 5%. In both
tissues, [Phe
]bombesin showed highest affinity,
and [SAP]bombesin relatively lower affinity. Thus these new
peptides are clearly agonists at the known bombesin receptors, but have
differing affinities.
Figure 6:
Luminometric assay of the response of Xenopus oocytes expressing the human GRP and NMB receptors to
[Phe]bombesin, [SAP]bombesin, and
[SAP]bombesin(5-14). Oocytes were co-injected with 5 ng
of human GRP or NMB receptor RNA transcribed from a cDNA encoding the
receptor and the calcium photoprotein aequorin(21) . After 24 h
the eggs were challenged with 10 nM concentration of ligand
and the light output was measured over 120 s in a luminometer. Results
show averages of three independent injected oocytes for each compound
and receptor. The overall lower responses seen in oocytes injected with
the NMB receptor reflect a smaller amount of injected transcript. The
relatively low response to NMB itself may reflect partial oxidation of
the peptide.
Figure 7: Displacement curves for GRP receptor selective assay (A) and NMB selective assay (B) for the peptides shown. Data shown is representative of 3-4 independent determinations.
The complexity of the bombesin-like peptide family is becoming apparent. Phylogenetic analysis of bombesin-like peptides identifies three subfamilies, the GRP family, the NMB family, and the BRP family (Fig. 1). The BRP family is composed of bombesin, ranatensin, and phyllolitorin, the bombesin-like peptides that occur in frog skin. Different species of frogs have different BRP's; B. orientalis have bombesin, R. pipiens have ranatensin, and P. sauvagei have phyllolitorin; but all have highly related prohormones. GRP and bombesin are clearly distinct peptides as frogs have both GRP and bombesin. Previous Northern blot analyses by our laboratory (14, 19) and Richter et al.(44, 45) has shown that in frogs, bombesin occurs at highest levels in skin, brain, and oocytes, while GRP occurs at highest levels in the GI tract(14) .
Our observation that
[Leu]phyllolitorin and
[Phe
]phyllolitorin are encoded by mRNAs differing
by only a single nucleotide led us to determine if there might be
multiple forms of bombesin also encoded by homologous mRNAs. As shown
in Fig. 2, in B. orientalis there are 3 forms
of bombesin encoded by 3 highly homologous mRNAs. The three forms of
bombesin are [Leu
]bombesin,
[Phe
]bombesin, and
[Ser
,Arg
,Phe
]bombesin.
These different peptides are produced by a surprisingly limited number
of nucleotide changes. It thus appears that most frogs will have
multiple forms of bombesin or bombesin-related peptides. In P. sauvagei, phyllolitorin occurs in both a Phe
form
and a Leu
form. In X. laevis,
Wechselberger et al.(45) showed the existence of a
Phe form of bombesin. As will be described elsewhere, (
)we
have also identified both Phe and Leu forms bombesin in Xenopus. Thus in B. orientalis, P. sauvagei, and X. laevis there are multiple forms of
bombesin. This conservation of multiple forms of bombesin suggests
there will be conserved unique physiologic forms for these multiple
peptides.
As would be expected given the high nucleotide homology
between the cDNAs, the three bombesin prohormones are highly
homologous. The [Phe]bombesin prohormone
contains an additional dibasic cleavage site creating the potential for
an additional processed peptide. Bombesin is cleaved from its precursor
at its amino terminus following Ser-Leu residues(44) . Ser-Leu
also occurs within the sequence of [SAP]bombesin. If this
second Ser-Leu motif is used for cleavage, then a peptide homologous to
mammalian NMB, [SAP]bombesin(5-14) with the structure
[Gln
-Arg
]neuromedin B would be
produced. Whether this cleavage actually takes place remains to be
determined.
The 3 forms of bombesin could arise by independent
genes, alternate splicing, or RNA editing. While RNA editing is
uncommon, because RNA editing is responsible for producing
[Leu]phyllolitorin and
[Phe
]phyllolitorin(20) , it is important
to consider that possibility. Amplification of genomic DNA followed by
sequence analysis, showed the presence of all 3 forms in genomic DNA at
approximately equal proportions. This suggests that the three forms of
bombesin arise from independent genes and not by alternate splicing or
RNA editing. The high homology between the three forms suggests a
relatively recent gene duplication or gene conversion event. This also
shows that the genes for bombesin do not have introns within the coding
region as do the genes for GRP and
NMB(16, 17, 46) . Alternate splicing of large
exons which include the entire amplified region cannot be ruled out,
although seems unlikely. The observation of the identical DNA sequence
in the genomic clones and the clones generated by RT-PCR would tend to
rule out the possibility of a PCR artifact generating the multiple
forms. This is further supported by the different distributions
discussed below and the confirmation of the presence of
[SAP]bombesin by tandem mass spectrometry.
To further rule
out the possibility that the multiple observed forms could be a PCR
artifact and to prove translation and processing of the SAP prohormone,
mass spectrometry was used to identify [SAP]bombesin in skin.
In these experiments, the skin extract was first separated by HPLC and
each fraction was analyzed by MALDI mass spectrometry to first identify
the correct mass (M 1642) and then by PSD and high
energy CID analysis to establish the amino acid sequence. In both
cases, the mass assignments and MS fragmentation data were consistent
with the expected [SAP]bombesin analog. The identification of
the existence of [SAP]bombesin in skin also confirms that SAP
is cleaved adjacent to the Ser-Leu residues at position 43,44 despite
the fact that the Arg residue at position 41 which is conserved in a
similar cleavage site in relaxin (47) is changed to a Gly in
the SAP prohormone. This loss of the Arg residue may, however, decrease
efficiency of processing, and could increase the presence of other
forms of the [SAP]bombesin prohormone.
The distribution of
the three forms of bombesin was examined by RT-PCR. As stated above,
previous Northern blot analyses by our laboratory (14, 19) and Richter et al.(44, 45) indicate that in frogs, bombesin occurs
at highest levels in skin, brain, and oocytes, while GRP occurs at
highest levels in GI tract(14) .
[Leu]Bombesin and [SAP]bombesin were
widely distributed. [Leu
]Bombesin was present in
all tissues examined except heart and liver. Notably,
[Leu
]bombesin was the predominant form of
bombesin present in oocytes. As will be reported elsewhere,
a Leu form of bombesin also predominates in Xenopus oocytes. Both [SAP]bombesin and
[Leu
]bombesin were present in lung. This is
significant given the findings of Aguayo et al.(48) and Sunday and co-workers (49, 50) that bombesin and phyllolitorin stimulate
lung development in mice. Thus a [SAP]bombesin or
[Leu
]bombesin homolog could be present in rodent
lung. [Phe
]Bombesin was found only in brain.
This is consistent with the observation that
[Phe
]phyllolitorin is the predominant form of
phyllolitorin in P. sauvagei brain (20) and a
Phe form of bombesin was isolated from Xenopus brain(45) . Thus in amphibian brain, a bombesin-related
peptide with Phe in the penultimate COOH-terminal position appears to
be frequently expressed.
The distribution within brain was examined
in dissected brain region. [Phe]Bombesin and
[Leu
]bombesin were found in all brain regions.
Interesting [SAP]bombesin was most abundant in pituitary and
spinal cord. Relatively little [Phe
]bombesin was
present in pituitary. The expression of a novel bombesin-like peptide
in pituitary is interesting given the many effects of bombesin-like
peptides that have been reported on pituitary
function(51, 52) .
Our laboratory has recently
cloned the apparent receptor for [Phe]bombesin
from B. orientalis brain(26) . This receptor
is expressed only in brain and has a K
for
[Phe
]bombesin of .2 nM, and defines a
new class (BB4) of bombesin receptors. The existence of this receptor
reinforces the potential importance of
[Phe
]bombesin as a brain neuropeptide.
Interestingly in rats, the amphibian ``Phe form''
bombesin-like peptide, [Phe
]phyllolitorin, has
potent effects on scratching behavior (53) raising the
possibility that mammals have homologous peptides. Specific receptors
for [Leu
]bombesin and [SAP]bombesin
likely exist and remain to be characterized.
If
[Phe]bombesin and [SAP]bombesin have
physiologic roles distinct from [Leu
]bombesin,
they should have different receptor affinities. As shown in Fig. 5and Fig. 6and Table 1,
[Phe
]bombesin and [SAP]bombesin are
agonists at both the GRP and NMB receptors. Neither of these peptides
had any significant affinity for the BRS-3
receptor(24, 25) , thus their structures give no hint
as to the identity of the BRS-3 ligand. [SAP]Bombesin has
relatively lower affinity for the known bombesin receptors; thus its
receptor may be quite distinct from the
[Phe
]bombesin receptor.
[SAP]Bombesin(5-14), an NMB agonist, could
potentially be produced by cleavage at the internal Ser-Leu motif
(position 47,48) of [SAP]bombesin. Whether this peptide is
produced in vivo in frog brain remains to be determined. A key
question remaining to be answered is if frogs have NMB or if, instead,
[Phe]bombesin or [SAP]bombesin perform
some of the same functions in frog brain as does NMB in mammalian
brain.
The existence of multiple forms of bombesin within a single
species of frog implies a more complicated physiologic role for
bombesin-like peptides than previously realized. This appears to be
generalizable as this occurs in multiple species of frogs. The distinct
distributions and affinities for each peptide further suggests distinct
physiologic roles. Given that the bombesin-related peptides diverged
from GRP and NMB prior to the vertebrate radiation (20) it is
quite likely that BRPs distinct from GRP will also exist in mammals,
and quite likely in multiple forms. Given the high affinity of
[Phe]bombesin both for its native receptor (26) and for the GRP and NMB receptors and given the prevalence
of [Phe]bombesin in amphibian brain, a BRP with Phe as the
penultimate residue may also exist in mammals and may mediate some of
the effects ascribed to bombesin-like peptides in mammals.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U49450 [GenBank]and U49451[GenBank].