The bombesin receptor subtypes have distinct G protein specificities

We used an in situ reconstitution assay to examine the receptor coupling to purified G protein alpha subunits by the bombesin receptor family, including gastrin-releasing peptide receptor (GRP-R), neuromedin B receptor (NMB-R), and bombesin receptor subtype 3 (BRS-3). Cells expressing GRP-R or NMB-R catalyzed the activation of squid retinal Galphaq and mouse Galphaq but not bovine retinal Galphat or bovine brain Galphai/o. The GRP-R- and NMB-R-catalyzed activations of Galphaq were dependent upon and enhanced by different betagamma dimers in the same rank order as follows: bovine brain betagamma > beta1gamma2 > beta1gamma1. Despite these qualitative similarities, GRP-R and NMB-R had distinct kinetic properties in receptor-G protein coupling. GRP-R had higher affinities for bovine brain betagamma, beta1gamma1, and beta1gamma2 and squid retinal Galphaq. In addition, GRP-R showed higher catalytic activity on squid Galphaq. Like GRP-R and NMB-R, BRS-3 did not catalyze GTPgammaS binding to Galphai/o or Galphat. However, BRS-3 showed little, if any, coupling with squid Galphaq but clearly activated mouse Galphaq. GRP-R and NMB-R catalyzed GTPgammaS binding to both squid and mouse Galphaq, with GRP-R activating squid Galphaq more effectively, and NMB-R also showed slight preference for squid Galphaq. These studies reveal that the structurally similar bombesin receptor subtypes, in particular BRS-3, possess distinct coupling preferences among members of the Galphaq family.     

Two distinct bombesin receptor subtypes are expressed and functional in human lung carcinoma cells.

Bombesin-like peptides have been implicated as autocrine growth factors influencing the pathogenesis and progression of some human lung carcinoma cells. To determine the pharmacologic and structural properties of the bombesin receptors expressed in human lung carcinoma cells, cDNA clones encoding a human gastrin-releasing peptide receptor (GRP-R) and a pharmacologically distinct neuromedin-B preferring bombesin-receptor (NMB-R) were isolated from a human small cell lung carcinoma cell line (NCI-H345). After expression in Xenopus oocytes, a GRP-R-specific antagonist was effective in blocking responses elicited from the cloned GRP-R, but not the NMB-R. Both GRP-R and NMB-R mRNA expression was detected at varying levels in a panel of human lung cancer cell lines. These results indicate heterogeneity of bombesin receptor subtypes exists in human lung carcinoma cells and should be considered in the design of bombesin receptor antagonists intended to inhibit tumor cell growth.     

Functional assay for GABA receptor subtypes of a cockroach giant interneuron.

gamma-Aminobutyric acid (GABA) receptors were examined in the cockroach central nervous system (CNS) using the single fiber-oil gap method applied to an identified giant interneuron. Short-lasting pressure application of 10 mM GABA developed a multiphasic response composed of a fast hyperpolarization followed by a transient depolarizing component and a stable hyperpolarization. This triphasic characteristic shape of the response was modified according to the dose of GABA injected or bath-applied and to the precise localization of the injection within the dendritic area. The transient depolarizing phase showed a negative reversal potential of -70 mV. Both hyperpolarizing phases reversed at a more negative level ranging to -80 mV. A positive shift of these values was caused by a decrease in external chloride concentration. Bath-application of 0.1 mM picrotoxin (Ptx) decreased the depolarizing phase which was progressively replaced by a stable hyperpolarization. The transient depolarizing component desensitized quickly and was the most sensitive phase to Ptx action. The Ptx-resistant response reversed at a mean value of -100 mV close to the equilibrium potential for potassium ions (EK+), suggesting that it was generated by a K(+)-channel coupled receptor. Although baclofen was unable to mimic the Ptx-resistant GABA response, the compound CGA 147823, known to bind with a high specificity to vertebrate GABAB receptors, has been successfully used to reproduce the Ptx-resistant GABA response. It is suggested that, in addition to GABA receptors linked to chloride channels, the insect CNS possesses GABA receptors sharing ionic characteristics of GABAB receptors especially those located in the vertebrate CNS, although they are insensitive to baclofen.     

Parallel bioassay of 27 bombesin-like peptides on 9 smooth muscle preparations. Structure-activity relationships and bombesin receptor subtypes

Thirteen natural bombesin-like peptides and 14 synthetic analogues were submitted to parallel bioassay on 9 smooth muscle preparations in order to determine their relative potency, in comparison to bombesin and litorin. The natural peptides of the bombesin subfamily showed a uniformly high or moderate potency on all preparations. However, synthetic bombesins of shorter chain length (hepta- and octapeptides) manifested a good potency only on the rat uterus preparation. Among the peptides of the litorin and phyllolitorin subfamilies, only litorin and ranatensin presented a full spectrum of potency, equalling or even surpassing that of bombesin. All other natural and synthetic members of the two subfamilies showed a sharply dissociated spectrum of potency on the different smooth muscle preparations. The only exception was the rat urinary bladder and, in part, the chicken intestine, on which the peptides displayed a uniformly high potency, comparable to, or even greater than that of bombesin. The present results help to explain structure/activity relationships and suggest the probable existence, in the periphery, of multiple bombesin receptor subtypes.     

Des-Met14]bombesin analogues function as small cell lung cancer bombesin receptor antagonists.

A series of bombesin (BN) analogues lacking the C-terminal methionine at the 14 position were evaluated as BN receptor antagonists. [D-Phe6]BN(6-13)amide inhibited specific 125I-GRP binding to lung cancer cell line NCI-H720 with an IC50 value of 12 nM. In contrast, [D-Phe6]BN(6-13)propylamide, butylamide and methylester were more potent with IC50 values of 3, 5 and 5 nM whereas [D-Phe6,Sta13]BN(6-13)amide was less potent with an IC50 value of 180 nM. [D-Phe6]BN(6-13)propylamide antagonized the ability of BN to elevate cytosolic Ca2+, whereas [D-Phe6]BN(6-13)butylamide was a partial agonist. In a small cell lung cancer (SCLC) growth assay, [D-Phe6]BN(6-13)propylamide inhibited colony formation. In summary, BN analogues which lack a C-terminal methionine may function as useful SCLC BN receptor antagonists.     

Syntheses and biological activities of potent bombesin receptor antagonists.

Bombesin receptor antagonists are potential therapeutic agents due to their ability to act as inhibitors of cellular proliferation. On the basis of our hypothesis concerning the mechanism of action of gastrin associating an activating enzyme to the receptor and on the results reported in the literature, we have synthesized bombesin analogs which have been modified in the C-terminal part. Potent bombesin receptor antagonists were obtained by replacement of Leu-13 with a statyl residue or with a residue bearing an hydroxyl group in place of the carbonyl function of Leu-13. Several inhibitors were able to recognize the bombesin receptor on rat pancreatic acini and antagonized bombesin stimulated amylase secretion in the nanomolar range. These compounds were also able to recognize the bombesin receptor and to inhibit [3H] thymidine incorporation in 3T3 cells with the same potency.     

Proposed loci of interaction between bombesin and its receptor.

The non-coding strand of the bombesin receptor gene, when 'translated' 5' to 3', contains an interrupted sequence of the 10 amino acids of bombesin. This finding forms the basis for proposing the points of contact between bombesin and its receptor as well as a partial conformation of the binding region of the receptor.     

G-protein-coupled receptors: the new dopamine receptor subtypes.

The family of genes encoding G-protein-coupled dopamine receptors continues to grow with the recent cloning of a fifth member. The availability of these clones has revolutionized the dopamine receptor field. Expression of individual dopamine receptors is permitting the detailed analysis of their pharmacology and coupling to second messenger systems, while probes based on the receptors' nucleotide sequences are being used to gain new insights into their tissue distribution and genetics.     

Solubilization and characterization of the chicken oocyte vitellogenin receptor.

This paper describes the biochemical characterization of the chicken oocyte plasma-membrane receptor for one of the major lipid-carrying yolk proteins, vitellogenin (VTG). The receptor was extracted from oocyte membranes with the non-ionic detergent octyl-beta-D-glucoside and visualized by ligand blotting, with 125I-VTG as a protein with an apparent Mr of 96000, under non-reducing conditions. It exhibited high affinity for native chicken VTG (Kd 2 X 10(-7) M) but was unable to bind VTG with reductively methylated lysine residues or phosvitin (the phosphoserine-rich intracellular cleavage product of VTG). Polyclonal antibodies to the 96 kDa protein inhibited VTG binding to the receptor and were able to precipitate functional VTG-receptor activity from oocyte-membrane detergent extracts with a concomitant removal of the 96 kDa protein. Antibodies directed against the mammalian receptor for low-density lipoprotein showed cross-reactivity with the chicken oocyte VTG receptor, raising the possibility that lipoprotein receptors in birds are structurally related to those in mammalian species.     

Distinguishing three subtypes of hematopoietic cells based on gene expression profiles using a support vector machine

Hematopoiesis is a complicated process involving a series of biological sub-processes that lead to the formation of various blood components. A widely accepted model of early hematopoiesis proceeds from long-term hematopoietic stem cells (LT-HSCs) to multipotent progenitors (MPPs) and then to lineage-committed progenitors. However, the molecular mechanisms of early hematopoiesis have not been fully characterized. In this study, we applied a computational strategy to identify the gene expression signatures distinguishing three types of closely related hematopoietic cells collected in recent studies: (1) hematopoietic stem cell/multipotent progenitor cells; (2) LT-HSCs; and (3) hematopoietic progenitor cells. Each cell in these cell types was represented by its gene expression profile among a total number of 20,475 genes. The expression features were analyzed by a Monte-Carlo Feature Selection (MCFS) method, resulting in a feature list. Then, the incremental feature selection (IFS) and a support vector machine (SVM) optimized with a sequential minimum optimization (SMO) algorithm were employed to access the optimal classifier with the highest Matthews correlation coefficient (MCC) value of 0.889, in which 6698 features were used to represent cells. In addition, through an updated program of MCFS method, seventeen decision rules can be obtained, which can classify the three cell types with an overall accuracy of 0.812. Using a literature review, both the rules and the top features used for building the optimal classifier were confirmed to be commonly used or potential biological markers for distinguishing the three cell types of HSPCs. This article is part of a Special Issue entitled: Accelerating Precision Medicine through Genetic and Genomic Big Data Analysis edited by Yudong Cai & Tao Huang.     

Characterization of the murine pancreatic receptor for gastrin releasing peptide and bombesin.

The murine pancreatic receptor for bombesin and gastrin releasing peptide (GRP) has been characterized. Analysis of the binding of 125I-GRP to membranes indicates a single class of sites (10(-13) mol/mg protein) with Kd of 43 pM. A 70 kDa membrane protein was cross-linked to 125I-GRP by bis(sulfosuccinimidyl) suberate; labeling was blocked by GRP, GRP (14-27), AcGRP(20-27), GRP(18-27), bombesin and ranatensin, was partially blocked by [Leu13 psi (CH2NH)Leu14]bombesin and was unaffected by GRP(21-27) and GRP(1-16). The IC50 values for the competitive displacement of 125I-GRP from intact membranes by these peptides were similar to those obtained by the cross-linking experiments showing that the 70 kDa protein is the GRP receptor. The GRP receptor is G-protein coupled; divalent cations are required for high-affinity binding and nonhydrolyzable GTP analogs decrease receptor affinity. In minced pancreas, GRP caused a dose-dependent increase in inositol phosphates implicating phospholipase C in signal transduction. We suggest that the murine pancreatic receptor for bombesin/GRP is a 70 kDa membrane protein, is associated with a G-protein and stimulates phosphatidylinositol turnover.     

des-Met carboxyl-terminally modified analogues of bombesin function as potent bombesin receptor antagonists, partial agonists, or agonists.

In the present study we examined the effect of carboxyl-terminal modifications of des-Met14-bombesin (Bn) on Bn receptor affinity in murine 3T3 cells, rat and guinea pig pancreatic acini, and the ability to initiate biologic responses by synthesizing 18 des-Met14-Bn(6-13) analogues. With guinea pig acini and 3T3 cells, affinity was affected by the chain length of the alkyl moiety (R) added to [D-Phe6]Bn(6-13)NH2R with relative potencies: propyl greater than ethyl greater than butyl = hexyl greater than heptyl greater than free amide, whereas in rat acini affinity was not increased by the chain length. In each cell system the affinity of the alkylamide was not increased by insertion of a phenyl group in the alkyl side chain, by making the analogue more neuromedin B-like or by addition of a reduced peptide bond. The affinity in each cell system was increased by additions of other electron releasing groups to the COOH-terminal carboxyl group such as [D-Phe6]Bn(6-13)ethyl or methyl ester, or hydrazide. In guinea pig pancreas and 3T3 cells, 12 analogues were antagonists, 1 a full and 5 partial agonists. In rat pancreas, 8 were antagonists, 5 full agonists, and 5 partial agonists. Potent antagonists in each cell system were the methyl and ethyl ester, hydrazide, and ethylamide analogues. In 3T3 cells or guinea pig pancreas, agonist activity of the alkylamide was critically dependent on the chain length, whereas with rat pancreatic Bn receptors any alkylamide longer than the ethylamide had agonist activity. In all three cell systems any alteration that made the alkylamide more neuromedin B-like caused agonist activity. These results demonstrate that the nature of the substitution on the carboxyl terminus of des-Met14-Bn analogues is critically important, not only for determining Bn receptor affinity, but also for determining the ability to initiate a biologic response. In contrast to previous studies, the present results demonstrate that the presence of the COOH-terminal amino acid in position 14 of Bn is not essential for initiating a biologic response. Several des-Met14-Bn analogues were potent partial agonists, whereas others such as the hydrazide or ethyl ester are very potent antagonists.     

Localization of cholecystokinin receptor subtypes in the endocine pancreas.

This study was undertaken to clarify the controversy in the literature about pancreatic localization of the cholecystokinin (CCK) CCK(A) and CCK(B) receptors. With antibodies used by other investigators, we first established their specificity by Western blotting, indirect immunofluorescence, and confocal microscopy with each antibody's peptide antigen. Co-localization assays between the CCK receptors and the pancreatic hormones insulin, glucagon, and somatostatin revealed that the CCK(A) RAbs 1122 and R1-2 recognized insulin and glucagon cells in rat, pig, and human pancreas but not in the somatostatin cells. Conversely, the three CCK(B) RAbs tested, 9262, 9491, and GR4, identified the somatostatin cells. Abs 9491 and GR4 occasionally co-localized with glucagon, a feature that never occurred with Ab 9262. Finally, the specificity of Ab 9262 for the pancreatic CCK(B) R was confirmed in six different species. It co-localized with somatostatin but never with glucagon in these species. Our data suggest the use of Abs 1122 and 9262 to specifically identify and localize pancreatic CCK(A) and CCK(B) receptors, respectively. Confusion in the literature may result from the lack of specificity of most antibodies used, as established in this study.     

Radioligand receptor assay for prolactin using chicken and turkey kidney membranes.

1. A sensitive and specific radioreceptor assay for measuring prolactin in tissue extracts, culture media and serum has been developed utilizing membrane fractions prepared from either chicken or turkey kidney, and an ovine prolactin standard and tracer. 2. Assay sensitivity was 1.0 +/- 0.1 ng per tube, 50% inhibition of binding occurred with 12.8 +/- 1.2 ng of unlabeled ovine prolactin standard, and intra- and interassay coefficients of variation were 3.9% and 8.8%, respectively. 3. The 47,800 g kidney membrane preparation, which yielded maximum specific binding of 20-40%, offered advantages over current methods including use of an easily available and inexpensive tissue, no need for pretreatment of the donor animal, a high yield of receptor protein, and a simplified method of membrane preparation.     

Multiple muscarinic receptor subtypes in the canine pulmonary circulation.

The vascular response to the muscarinic receptor agonist acetylcholine (ACh) in the presence of selected antagonists was examined in the isolated blood-perfused canine left lower lung lobe under conditions of normal (resting) and elevated vascular tone. At normal vascular tone, ACh (1-5 mumol) produced a dose-dependent increase in pulmonary arterial pressure (Ppa), total pulmonary vascular resistance (PVR), and downstream resistance (Rds) without altering upstream resistance (Rus). Pirenzepine (50 and 100 nM), the prototype M1-selective antagonist, and gallamine, an M2-selective antagonist, as well as atropine (50 nM) and secoverine (100 nM), nonselective antagonists, attenuated (P less than 0.05) the ACh-induced increase in Ppa and Rds. With elevated vascular tone induced by serotonin infusion, ACh produced a dose-dependent increase in Ppa in 19 of 25 lobes, although Rus decreased while Rds increased in all lobes. At high vascular tone, pirenzepine or gallamine attenuated the ACh-induced increase in Rds, whereas Rus was not affected. Secoverine and atropine antagonized ACh-induced increases in both Rds and Rus. The pA2 values (i.e., the negative log antagonist concentration requiring a doubling of ACh dose for an equivalent increase in Rds) for gallamine, pirenzepine, secoverine, and atropine were 6.1 +/- 0.1, 7.4 +/- 0.1, 8.3 +/- 0.2, and 10.2 +/- 0.3, respectively. These results suggest that 1) ACh increases PVR in the dog by constricting the venous segments (downstream) of the pulmonary circulation via activation of pulmonary vascular muscarinic receptors under conditions of both normal and elevated vascular tone, 2) both M1- and non-M1-muscarinic receptor subtypes appear to participate in mediating the ACh-induced increase in Rds, and 3) ACh moderately relaxes the upstream (arterial) vessels, especially under conditions of elevated tone.     

The receptor for yolk lipoprotein deposition in the chicken oocyte.

The final rapid growth phase of the chicken oocyte is characterized by massive uptake of hepatically synthesized yolk precursor proteins from the plasma. The two major yolk-forming components, very low density lipoprotein (VLDL) and vitellogenin (VTG), have been shown to interact with a 95-kDa protein present in detergent extracts of ovarian membranes; this protein is absent in hens of a mutant nonlaying chicken strain (Nimpf, J., Radosavljevic, M., and Schneider, W. J. (1989) J. Biol. Chem. 264, 1393-1398). Here, we have purified the 95-kDa protein by ligand and immunoaffinity chromatography and demonstrated its role in receptor-mediated endocytosis by ultrastructural immunolocalization, structural, and functional studies. The receptor was visualized exclusively in the oocyte proper and was absent from somatic cells, in agreement with the previously reported expression of two different lipoprotein receptors in somatic cells and oocytes, respectively, of laying hens (Hayashi, K., Nimpf, J., and Schneider, W. J. (1989) J. Biol. Chem. 264, 3131-3139). Amino acid sequences of tryptic fragments of the oocyte receptor were obtained, and its kinship to somatic low density lipoprotein receptors was confirmed through the demonstration of sequence conservation in three characteristic domains. In particular, the chicken receptor's internalization sequence, Phe-Asp-Asn-Pro-Val-Tyr, is identical with that in low density lipoprotein receptors from mammals as well as Xenopus laevis. The ligand-binding properties, specificity, and kinetic parameters of the oocyte receptor were characterized in filtration assays employing pure ligands and receptor. In conjunction with ligand-blotting experiments following limited protease digestion of the receptor, the binding assay data suggest that VTG recognizes a substructure of the VLDL-binding site. These studies establish that a cell-specific receptor mediates the endocytosis of VTG and VLDL into growing chicken oocytes and thus possibly plays a key role in control of oocyte growth.     

Metabolic stability and tumor inhibition of bombesin/GRP receptor antagonists.

Small cell lung cancers (SCLC) synthesize and secrete bombesin/gastrin releasing peptide (BN/GRP). The autocrine growth cycle of BN/GRP in SCLC can be disrupted by BN/GRP receptor antagonists such as [Psi13,14]BN. Here several BN analogues were solid-phase synthesized and incubated with intact SCLC cells at 37 degrees C in RPMI medium in a time-course fashion (0-1080 minutes) to determine enzymatic stability. The proteolytic stability of the compounds was determined by subsequent HPLC analysis. The metabolic half-life ranged from 154 minutes to 1388 minutes for the six analogues studied. [Psi13,14]BN was found to be very stable to metabolic enzymes (T1/2 = 646 mm) and also inhibited SCLC xenograft formation in vivo in a dose-dependent manner. When [Psi13,14]BN was incubated with NCI-H345 cells, it inhibited 125I-GRP binding with an IC50 value of 30 nM. These data suggest that BN/GRP receptor antagonists such as [Psi13,14]BN may be useful for the treatment of SCLC.     

An attempt to analyze various thyroid stimulators by the receptor assay using hTSH radioimmunoassay.

In an attempt to analyze thyroid stimulators in serum we developed an assay procedure using hTSH radioimmunoassay (RIA) in combination with receptor competition. The principle of this method is the determination by RIA of hTSH displaced by other thyroid stimulators from a thyroidal receptor preparation which previously bound unlabelled hTSH. Practically 4 microunits of hTSH were bound with human or bovine receptor, and then hTSH displaced by addition of test serum (0.1 ml) or samples dissolved in serum (0.1 ml) was measured by RIA. This assay can determine the thyroid stimulators other than hTSH in serum that has the displacement activity of 0.5-4.0 microunits of hTSH in the useful range, such as mU/ml level of bovine TSH or rat TSH. Cholera toxin that has the thyroid stimulating activity like TSH also showed the displacement of the bound hTSH. This assay is not applicable for the human serum with more than 5 microunits/ml of TSH, because the assay value is over estimated by the free hTSH derived from the test serum. On the other hand, eighteen sera with high LATS activity and 42 sera with negative LATS activity from patients with untreated hyperthyroidism did not show any displacement. This might be due to the lower binding activity of LATS with hTSH receptor or the lower sensitivity of this assay method. Although it is difficult to use this assay clinically because of its low sensitivity, increased TSH in animal serum can be determined by this assay. The principle of this method may be also useful for examining the receptor binding of other peptide hormone that can be determined by an RIA method.