Bombesin-like peptide receptor gene expression,regulation, and function in fetal murine lung

Bombesin-peptide (BLP) immunoreactivity occurs at high levels in fetal lung. Previous studies showed that bombesin promotes fetal lung development. To test the hypothesis that such effects are mediated by known mammalian bombesin receptors [gastrin-releasing peptide (GRP)/bombesin-preferring receptor (GRPR), neuromedin B (NMB) receptor (NMBR), and the orphan bombesin receptor subtype-3 (BRS-3)], we analyzed the ontogeny of GRPR, NMBR, and BRS-3 gene expression in mouse lung. We examined the regulation of these three genes by dexamethasone and bombesin, which modulate lung development. Using incorporation of [3H]thymidine and [3H]choline, we then assessed whether GRP, NMB, and Leu8-phyllolitorin modulate lung growth and maturation in fetal lung explants. GRPR gene expression was detected predominantly in utero, whereas NMBR and BRS-3 genes were expressed from embryonic days 13-16 and on multiple postnatal days. All three mRNAs are present in airway epithelium and mesenchymal cells but occur in different relative patterns. These genes were regulated differently. Dexamethasone and bombesin increased GRPR mRNA, bombesin downregulated NMBR, and neither agent affected BRS-3. GRP increased incorporation of [3H]thymidine and [3H]choline in explants, whereas NMB induced cell proliferation and Leu8-phyllolitorin yielded variable results. Cumulative data suggest the involvement of multiple BLP receptors, including novel molecules, and argue against simple functional redundancy within this gene family during lung development.     

Molecular basis for selectivity of high affinity peptide antagonists for the gastrin-releasing peptide receptor.

Few gastrointestinal hormones/neurotransmitters have high affinity peptide receptor antagonists, and little is known about the molecular basis of their selectivity or affinity. The receptor mediating the action of the mammalian bombesin (Bn) peptide, gastrin-releasing peptide receptor (GRPR), is an exception, because numerous classes of peptide antagonists are described. To investigate the molecular basis for their high affinity for the GRPR, two classes of peptide antagonists, a statine analogue, JMV594 ([d-Phe(6),Stat(13)]Bn(6-14)), and a pseudopeptide analogue, JMV641 (d-Phe-Gln-Trp-Ala-Val-Gly-His-Leupsi(CHOH-CH(2))-(CH(2))(2)-CH(3)), were studied. Each had high affinity for the GRPR and >3,000-fold selectivity for GRPR over the closely related neuromedin B receptor (NMBR). To investigate the basis for this, we used a chimeric receptor approach to make both GRPR loss of affinity and NMBR gain of affinity chimeras and a site-directed mutagenesis approach. Chimeric or mutated receptors were transiently expressed in Balb/c 3T3. Only substitution of the fourth extracellular (EC) domain of the GRPR by the comparable NMBR domain markedly decreased the affinity for both antagonists. Substituting the fourth EC domain of NMBR into the GRPR resulted in a 300-fold gain in affinity for JMV594 and an 11-fold gain for JMV641. Each of the 11 amino acid differences between the GRPR and NMBR in this domain were exchanged. The substitutions of Thr(297) in GRPR by Pro from the comparable position in NMBR, Phe(302) by Met, and Ser(305) by Thr decreased the affinity of each antagonist. Simultaneous replacement of Thr(297), Phe(302), and Ser(305) in GRPR by the three comparable NMBR amino acids caused a 500-fold decrease in affinity for both antagonists. Replacing the comparable three amino acids in NMBR by those from GRPR caused a gain in affinity for each antagonist. Receptor modeling showed that each of these three amino acids faced inward and was within 5 A of the putative binding pocket. These results demonstrate that differences in the fourth EC domain of the mammalian Bn receptors are responsible for the selectivity of these two peptide antagonists. They demonstrate that Thr(297), Phe(302), and Ser(305) of the fourth EC domain of GRPR are the critical residues for determining GRPR selectivity and suggest that both receptor-ligand cation-pi interactions and hydrogen bonding are important for their high affinity interaction.     

Tyrosine 220 in the 5th transmembrane domain of the neuromedin B receptor is critical for the high selectivity of the peptoid antagonist PD168368

Peptoid antagonists are increasingly being described for G protein-coupled receptors; however, little is known about the molecular basis of their binding. Recently, the peptoid PD168368 was found to be a potent selective neuromedin B receptor (NMBR) antagonist. To investigate the molecular basis for its selectivity for the NMBR over the closely related receptor for gastrin-releasing peptide (GRPR), we used a chimeric receptor approach and a site-directed mutagenesis approach. Mutated receptors were transiently expressed in Balb 3T3. The extracellular domains of the NMBR were not important for the selectivity of PD168368. However, substitution of the 5th upper transmembrane domain (uTM5) of the NMBR by the comparable GRPR domains decreased the affinity 16-fold. When the reverse study was performed by substituting the uTM5 of NMBR into the GRPR, a 9-fold increase in affinity occurred. Each of the 4 amino acids that differed between NMBR and GRPR in the uTM5 region were exchanged, but only the substitution of Phe(220) for Tyr in the NMBR caused a decrease in affinity. When the reverse study was performed to attempt to demonstrate a gain of affinity in the GRPR, the substitution of Tyr(219) for Phe caused an increase in affinity. These results suggest that the hydroxyl group of Tyr(220) in uTM5 of NMBR plays a critical role for high selectivity of PD168368 for NMBR over GRPR. Receptor and ligand modeling suggests that the hydroxyl of the Tyr(220) interacts with nitrophenyl group of PD168368 likely primarily by hydrogen bonding. This result shows the selectivity of the peptoid PD168368, similar to that reported for numerous non-peptide analogues with other G protein-coupled receptors, is primarily dependent on interaction with transmembrane amino acids.     

Targeting gastrin-releasing peptide receptors of prostate cancer cells for photodynamic therapy with a phthalocyanine-bombesin conjugate

Sulfonated aluminum phthalocyanines (AlPcS) are potent photosensitizers for the photodynamic therapy (PDT) of cancer. In this study we evaluate the possibility to improve the efficacy of AlPcS-PDT for prostate cancer by targeting tetrasulfonated aluminum phthalocyanines (AlPcS(4)) to the gastrin-releasing peptide receptor (GRPR) through coupling to bombesin. A mono-carbohexyl derivative of AlPcS(4) is attached to 8-Aoc-bombesin(7-14)NH(2) via an amide bridge to yield a bombesin-AlPcS(4) conjugate linked by a C-14 spacer chain. The conjugate is characterized by mass spectroscopy and shown to bind to the GRPR with a relative binding affinity (RBA) of 2.3, taking bombesin (RBA=100) as unity. The in vitro photodynamic efficacy of the conjugate against PC-3 human prostate cancer cells is improved by a factor 2.5 over the non-conjugated mono-carbohexyl derivative of AlPcS(4).     

Bombesin-like peptide and receptors in lung injury models: diverse gene expression, similar function

We previously demonstrated that bombesin-like peptide (BLP) mediates lung injury in premature infants with bronchopulmonary dysplasia (BPD). We now investigate gene expression and function of BLP (gastrin-releasing peptide, GRP) and BLP-receptors (GRP-R and BRS-3) in lung from two baboon BPD models. In the "interrupted gestation model," only GRP mRNA was up-regulated. In the "hyperoxic model," GRP-R mRNA was up-regulated. In lung explants from O2-treated animals, all BPD animals responded to 1nM bombesin, whereas non-BPD animals did not; the opposite effect was observed with a BLP blocking antibody. Cumulatively, these observations suggest that novel BLPs and/or BLP receptors are likely to be implicated in the pathogenesis of BPD.     

Concomitant vascular GRP-receptor and VEGF-receptor expression in human tumors: molecular basis for dual targeting of tumoral vasculature

Gastrin-releasing peptide (GRP) and GRP receptors (GRPR) play a role in tumor angiogenesis. Recently, GRPR were found to be frequently expressed in the vasculature of a large variety of human cancers. Here, we characterize these GRPR by comparing the vascular GRPR expression and localization in a selection of human cancers with that of an established biological marker of neoangiogenesis, the vascular endothelial growth factor (VEGF) receptor. In vitro quantitative receptor autoradiography was performed in parallel for GRPR and VEGF receptors (VEGFR) in 32 human tumors of various origins, using ¹²⁵I-Tyr-bombesin and ¹²⁵I-VEGF₁₆₅ as radioligands, respectively. Moreover, VEGFR-2 was evaluated immunohistochemically. All tumors expressed GRPR and VEGFR in their vascular system. VEGFR were expressed in the endothelium in the majority of the vessels. GRPR were expressed in a subpopulation of vessels, preferably in their muscular coat. The vessels expressing GRPR were all VEGFR-positive whereas the VEGFR-expressing vessels were not all GRPR-positive. GRPR expressing vessels were found immunohistochemically to co-express VEGFR-2. Remarkably, the density of vascular GRPR was much higher than that of VEGFR. The concomitant expression of GRPR with VEGFR appears to be a frequent phenomenon in many human cancers. The GRPR, localized and expressed in extremely high density in a subgroup of vessels, may function as target for antiangiogenic tumor therapy or angiodestructive targeted radiotherapy with radiolabeled bombesin analogs alone, or preferably together with VEGFR targeted therapy.     

Gastrin-releasing peptide (GRP) in the ovine uterus: regulation by interferon tau and progesterone

Gastrin-releasing peptide (GRP) is abundantly expressed by endometrial glands of the ovine uterus and processed into different bioactive peptides, including GRP1-27, GRP18-27, and a C-terminus, that affect cell proliferation and migration. However, little information is available concerning the hormonal regulation of endometrial GRP and expression of GRP receptors in the ovine endometrium and conceptus. These studies determined the effects of pregnancy, progesterone (P4), interferon tau (IFNT), placental lactogen (CSH1), and growth hormone (GH) on expression of GRP in the endometrium and GRP receptors (GRPR, NMBR, BRS3) in the endometrium, conceptus, and placenta. In pregnant ewes, GRP mRNA and protein were first detected predominantly in endometrial glands after Day 10 and were abundant from Days 18 through 120 of gestation. Treatment with IFNT and progesterone but not CSH1 or GH stimulated GRP expression in the endometrial glands. Western blot analyses identified proGRP in uterine luminal fluid and allantoic fluid from Day 80 unilateral pregnant ewes but not in uterine luminal fluid of either cyclic or early pregnant ewes. GRPR mRNA was very low in the Day 18 conceptus and undetectable in the endometrium and placenta; NMBR and BRS3 mRNAs were undetectable in ovine uteroplacental tissues. Collectively, the present studies validate GRP as a novel IFNT-stimulated gene in the glands of the ovine uterus, revealed that IFNT induction of GRP is dependent on P4, and found that exposure of the ovine uterus to P4 for 20 days induces GRP expression in endometrial glands.     

Evaluation of a technetium-99m labeled bombesin homodimer for GRPR imaging in prostate cancer

Multimerization of peptides can improve the binding characteristics of the tracer by increasing local ligand concentration and decreasing dissociation kinetics. In this study, a new bombesin homodimer was developed based on an ε-aminocaproic acid-bombesin(7–14) (Aca-bombesin(7–14)) fragment, which has been studied for targeting the gastrin-releasing peptide receptor (GRPR) in prostate cancer. The bombesin homodimer was conjugated to 6-hydrazinopyridine-3-carboxylic acid (HYNIC) and labeled with ^99mTc for SPECT imaging. The in vitro binding affinity to GRPR, cell uptake, internalization and efflux kinetics of the radiolabeled bombesin dimer were investigated in the GRPR-expressing human prostate cancer cell line PC-3. Biodistribution and the GRPR-targeting potential were evaluated in PC-3 tumor-bearing athymic nude mice. When compared with the bombesin monomer, the binding affinity of the bombesin dimer is about ten times lower. However, the ^99mTc labeled bombesin dimer showed a three times higher cellular uptake at 4 h after incubation, but similar internalization and efflux characters in vitro. Tumor uptake and in vivo pharmacokinetics in PC-3 tumor-bearing mice were comparable. The tumor was visible on the dynamic images in the first hour and could be clearly distinguished from non-targeted tissues on the static images after 4 h. The GRPR-targeting ability of the ^99mTc labeled bombesin dimer was proven in vitro and in vivo. This bombesin homodimer provides a good starting point for further studies on enhancing the tumor targeting activity of bombesin multimers.