The immune response toward beta-adrenergic ligands and their receptors. VIII. Extensive diversity of VH and VL genes encoding anti-alprenolol antibodies

The V region genes (VH and VL) used in the immune response of BALB/c mice to alprenolol, a synthetic beta-adrenergic ligand, were examined by Southern blot and nucleotide sequence analyses. Fourteen anti-alprenolol hybridomas utilize 10 different combinations of six Vk, one V lambda, eight VH, three JK, one J lambda, and three JH genes. In addition to the combinatorial association, somatic mutations and junctional variation of assembled genes further contribute to diversity of the anti-alprenolol response. Although differing both in length and structure, the five H-chain third complementarity-determining region analyzed contain several acidic residues. Neither V gene utilization, nor H-chain third complementarity-determining-region structure can be simply correlated with affinity of the antibodies for the ligand. The anti-alprenolol V genes were compared with the corresponding sequences of unrelated antibodies. Antibody 37A4 shares a VH gene with anti-(Glu60Ala30Tyr10)n random terpolymer and anti-nitrophenyl antibodies, and a Vk gene with two anti-oxazolone antibodies. Antibodies 14C3 and 17C1 use the same germ-line VH and Vk genes as do anti-anti-idiotypic antibodies of the (Glu60Ala30Tyr10) system. These data demonstrate the genetic diversity of the antibody response to alprenolol, and illustrate the extensive flexibility of the immune system.     

Functional expression of the human serotonin 5-HT1A receptor in Escherichia coli. Ligand binding properties and interaction with recombinant G protein alpha-subunits.

Signaling through serotonin 5-HT1A receptors involves multiple pathways. We have investigated the functional coupling of the human 5-HT1A receptor to different G proteins using an in vitro reconstitution system based on the expression of recombinant receptor (r5-HT1A) and G alpha-subunits (rG alpha) in Escherichia coli. The r5-HT1A receptor was expressed by insertion in a vector allowing its active expression in E. coli inner membranes. Binding of the selective agonist [3H] +/- 8-hydroxy-(2-N-dipropylamine)tetralin ([3H]8-OH-DPAT) to intact bacteria or E. coli membranes was saturable with a KD of approximately 8 nM and an average of 120 sites/bacterium. Binding properties of several serotoninergic ligands to r5-HT1A receptors were comparable with those measured in mammalian cells. Incubation of rG alpha.beta gamma with E. coli membranes resulted in high affinity agonist [3H]8-OH-DPAT binding (KD = 0.7 nM) and titration with a panel of rG alpha subtypes showed the order of potency: rGi alpha-3 greater than rGi alpha-2 greater than rGi alpha-1 much greater than rGo alpha, while rGs alpha appeared incapable of interacting with 5-HT1A receptors. Moreover, agonist-mediated enhancement of [35S]guanosine 5'-O-(3-thiotriphosphate) binding to rGi alpha confirmed the achievement of the functional interaction between receptor and G proteins. Our findings are in agreement with the in vivo ability of 5-HT1A receptors to activate Gi alpha related to adenylyl cyclase inhibition or K+ channel activation, but do not support previously reported adenylyl cyclase stimulation through interaction with Gs alpha.     

Coupling of ETB Endothelin Receptor to Mitogen-Activated Protein Kinase Stimulation and DNA Synthesis in Primary Cultures of Rat Astrocytes

Abstract: Astrocytes have been shown to express endothelin (ET) receptors functionally coupled, via different heterotrimeric G proteins, to several intracellular pathways. To assess the relative contribution of each subtype in the astrocytic responses to ET-1, effects of BQ123, an antagonist selective for the ET receptor subtype A (ET_A-R), and IRL1620, an agonist selective for the ET receptor subtype B (ET_B-R), were investigated in primary cultures of rat astrocytes. Binding experiments indicated that the ET_B-R is the predominant subtype in these cells. Inhibition of forskolin-stimulated cyclic AMP production was observed under ET_B-R stimulation. Bordetella pertussis toxin (PTX) pretreatment completely abolished this effect, indicating that this pathway is coupled to the ET_B-R via G_i protein. Increases of tyrosine phosphorylation of cellular proteins, stimulation of mitogen-activated protein kinase (MAPK), and DNA synthesis were also found to be mediated by the ET_B-R, but through PTX-insensitive G protein. IRL1620-induced MAPK activation involved the adapter proteins Shc and Grb2 and the serine/threonine kinase Raf-1. This study reveals that the various effects of ET-1 in astrocytes are mediated by the ET_B-R, which couples to multiple signaling pathways including the MAPK cascade.     

Monoclonal antibodies to protein kinase C gamma. Functional relationship between epitopes and cofactor binding sites

Four monoclonal antibodies (mAbs), derived from high-responder Biozzi mice immunized with purified protein kinase C, were selected by ELISA and further characterized by immunoblot: 3G12, 5A2, 36G9 are of isotype gamma 1, kappa and 15G4 is of isotype gamma 2b, kappa. Competition analysis between 15G4 and the three other mAbs showed that 15G4 and 3G12 are directed against either the same or overlapping epitope(s). All four mAbs are specific for the bovine gamma isoform of protein kinase C and cross-react with protein kinase C gamma from a variety of animal species. Immunoblot analysis of protein kinase C tryptic fragments revealed that the mAbs recognize the regulatory domain and not the catalytic domain. Two of the mAbs, 36G9 and 5A2, inhibit protein kinase C gamma cofactor-dependent activity (80% and 50% respectively). Consistent with the epitope mapping, none of mAbs inhibit the cofactor-independent catalytic activity of protein kinase C gamma. Competition analysis between these mAbs and phosphatidylserine, 12-O-tetradecanoylphorbol 13-acetate and Ca2+ showed that 36G9 and 5A2 block cofactor binding to protein kinase C gamma. These four mAbs thus interact with at least three distinct epitopes in the regulatory domain of protein kinase C gamma.     

Cholecystokinin octa- and tetrapeptide degradation by synaptic membranes. II. Solubilization and separation of membrane-bound CCK-8 cleaving enzymes

Solubilization of rat synaptic membranes by Triton X-100, followed by DEAE-cellulose chromatography allowed the identification of different CCK-8 cleaving enzymes. The first one (in the order of elution) removed the N-terminal aspartic acid residue of CCK-8 and was active on L-aspartic acid beta naphtylamide, suggesting that a corresponded to an aminopeptidase A. Two aminopeptidases of broad specificity hydrolyzed sequentially all the peptide bonds of CCK-8 as far as the release of free tryptophan. The removal of the sulfated tyrosine residue of CCK-8 occurred at a slower rate than that of the unsulfated residue. Another peptidase converted CCK-8 into its C-terminal heptapeptide. This enzyme had a lower affinity for the sulfated octapeptide in comparison with the unsulfated form (app Km of respectively 180 and 40 muM). The CCK-7 generating proteases displayed a moderate regional variation in five rat brain areas, with the highest activity in olfactory bulbs membranes and the lowest in cerebellar membranes. This distribution followed (with a lower amplitude) that of the CCK receptors.     

Stability of isoproterenol bound to cyanogen bromide-activated agarose.

The chemical stability and release of isoproterenol bound by diazotation to an insoluble agarose matrix has been investigated. It is demonstrated, by a double labeling procedure ([³H]isoproterenol and [¹⁴C]-spacer arm), that the bound ligand is readily released in a soluble form. This occurs primarily through a chemical hydrolysis of the arm-linked ligand from the cyanogen bromide-activated agarose, and can be observed under extensive washing procedures as well as under more “physiological” incubation conditions. The instability of the agarose-arm linkage should lead to a critical analysis of physiological effects obtained with agarose bound hormones in vitro.