Insulin induces alpha1B-adrenergic receptor phosphorylation and desensitization

The ability of insulin to induce alpha1B-adrenoceptor phosphorylation and desensitization was tested in two model systems: rat-1 cells that stably express alpha1B-adrenoceptors, through transfection, and endogenously express insulin receptors and DDT1 MF2 cells that endogenously express both receptors. Insulin induced concentration-dependent increases in the phosphorylation state of the adrenergic receptors in the two models with similar EC50 values (0.5-2 nM). The effect was rapid in the two systems but it was sustained in rat-1 cells and transient in DDT1 MF2 cells. In both cell lines, the insulin-mediated phosphorylation of alpha1B-adrenoceptors was blocked by wortmannin and LY 294002, and by staurosporine and bisindolylmaleimide I, indicating that the effect involved phosphoinositide 3-kinase and protein kinase C activities. The adrenoceptor phosphorylation induced by insulin was associated to desensitization as evidences by a diminished elevation of intracellular calcium in response to noradrenaline. Inhibitors of phosphoinositide 3-kinase and protein kinase C blocked the functional desensitization induced by insulin.     

Structure-activity relationship of synthetic truncated analogues of vasoactive intestinal peptide (VIP): an enhancement in the activity by a substitution with arginine

In order to develop potent shortened analogues of vasoactive intestinal peptide (VIP), the structure-activity relationship of C-terminally truncated analogues of VIP was investigated by examining the binding activity to rat lung VIP receptors and relaxation of smooth muscle in isolated mouse stomach. VIP(1-27) showed VIP receptor binding activity comparable to that of VIP but the activity of VIP(1-26) was reduced to one-third of VIP. The receptor binding activity of VIP(1-26) to VIP(1-23) was reduced in proportion to the decrease in amino acid residues. There was a significant correlation between the number of amino acid residues and VIP receptor binding activities of VIP and its C-terminally truncated analogues. VIP(1-22) and VIP(1-21) exhibited little binding activity even at high concentrations, suggesting the requisite of 23 amino acid residues as the minimal essential sequence for the conservation of VIP receptor binding activity. The chemical modification of VIP(1-23) generated a potent analogue, [Arg(15, 20, 21), Leu(17)]-VIP(1-23), that displayed a 22-fold higher receptor binding activity and 1.6-fold more potent relaxation of mouse stomach than VIP(1-23) did. In conclusion, it was shown that [Arg(15, 20, 21), Leu(17)]-VIP(1-23) could be a relatively potent and stable agonist of VIP receptors. The present study has provided further insight into the structure-activity relationship of VIP to generate novel shortened VIP analogues having a high affinity to VIP receptors and potent pharmacological activity.     

RGS8 expression in developing cerebellar Purkinje cells

The regulators of G protein signaling (RGS) proteins modulate heterotrimeric G protein signaling. RGS8 belongs to B/R4 subfamily of RGS proteins and is specifically expressed in Purkinje cells of adult cerebellum. Here, to examine the expression of RGS8 mRNA in developing cerebellum, we performed in situ hybridization. Apparent signals for expression of RGS8 mRNA were first detected on day 9 after birth, then RGS8 mRNA expression in Purkinje cells increased up to day 21, and its levels decreased to some extent in adult Purkinje cells. We also studied the expression of RGS7, which is expressed in Golgi cells in the granule cell layer of adult cerebellum. The expression of RGS7 mRNA was recognized in 7 day neonatal cerebellum. When examined with anti-RGS8 antibody, the RGS8 protein was already excluded from nucleus on day 9, and was distributed in cell body and dendrites in differentiating Purkinje cells of 14 day neonates.     

Overexpression of hepatocyte growth factor receptor in renal carcinoma cells indirectly stimulates tumor growth in vivo

We examined the role of increased expression of HGFR kinase in in vivo growth of renal carcinoma. Human renal carcinoma cell line, ACHN cells, was transfected with plasmid encoding wild-type HGFR gene to generate cell lines with increased HGFR protein. ACHN cells with elevated HGFR expression, denoted clones 8 and 10, respectively, showed higher basal kinase activities of HGFR and PI3-kinase than those of empty-vector (mock)-transfected cells. Clone 8 and 10 cells grew similar to mock cells in culture. In mice, tumors of these clones grew more rapidly than those of mock cells. Microvessel density of clone 8 or 10 tumors was higher than that of mock tumors. Clone 8 and 10 cells secreted vascular endothelial growth factor-A (VEGF-A) more than mock cells and the secretion was PI3-kinase inhibitor, LY294002-sensitive. Anti-VEGF-A neutralizing antibody significantly inhibited tumor growth of clones 8 and 10 in mice. These results indicate for the first time that overexpression of HGFR tyrosine kinase in renal carcinoma cells participates in rapid tumor growth in vivo.     

Expression of adrenomedullin and its receptor by chondrocyte phenotype cells

For clarifying a process of de-differentiation in culturing chondrocytes, the present study was undertaken to investigate the secretion of adrenomedullin (AM) by chondrocyte phenotype cells and whether or not AM effects this proliferation in a cAMP-dependent fashion. Chondrocyte phenotype cells expressed AM and the AM receptor, and secreted high concentration of AM into the culture medium. When added to cultures, AM increased the intracellular cAMP level and decreased the number of these cells in a similar concentration-dependent fashion. Addition of forskolin and dibutyryl-cAMP caused a significant decrease in the number of these cells. Furthermore, the effect of AM was inhibited by a cAMP-dependent protein kinase A inhibitor (H89). The present findings indicate that AM has an autocrine/paracrine type of anti-proliferative effect on these cells mediated via a cAMP-dependent pathway and raise the possibility that AM plays a role in the local modulation of a process of de-differentiation by culturing chondrocyte phenotype cells.     

Interaction of soluble CD4 with the chemokine receptor CCR5

The chemokine receptor CCR5 is constitutively associated with the T cell co-receptor CD4 in plasma cell membranes. The CD4-CCR5 complex exhibits distinct binding properties for macrophage inflammatory protein 1beta (MIP-1beta) and enhanced G-protein signaling as compared with those of CCR5 alone. Here we report that recombinant soluble CD4, when refolded into its dimeric form, allosterically modulates CCR5 and decreases the affinity for its natural ligand MIP-1beta. Monomeric soluble CD4 had little inhibitory effect on CCR5. In contrast, the two-domain amino-terminal fragment of soluble CD4 was able to completely inhibit the interaction of CCR5 with MIP-1beta. Thus, we suggest that various conformational states of CD4 exist, which differ markedly with regard to inhibiting the interaction of CCR5 with its ligand MIP-1beta. R5-tropic HIV-1 glycoprotein 120, but not interleukin-16, the natural agonist, or X4-tropic glycoprotein 120, inhibited MIP-1beta binding to CCR5 in the presence of monomeric and dimeric soluble CD4.     

Biochemical and functional properties of the full-length cation-dependent mannose 6-phosphate receptor expressed in Pichia pastoris

A glycosylation-deficient, full-length cation-dependent mannose 6-phosphate receptor (CD-MPR) containing a yeast signal sequence was expressed in Pichia pastoris using the constitutive promoter of the PGAP gene. The membrane-bound receptor was solubilized using detergents and purified by pentamannosyl phosphate-agarose affinity chromatography. Equilibrium binding studies identified a binding affinity of 2 nM for the lysosomal enzyme, beta-glucuronidase. To probe the linkage specificity of the recombinant CD-MPR, inhibition binding studies were conducted using non-phosphorylated oligomannoses which demonstrated that Manalpha1,2Man exhibits a 4-fold higher inhibition than Manalpha1,3Man and Manalpha1,6Man. The receptor was capable of associating into oligomeric forms and enzymatic deglycosylation revealed the presence of high-mannose sugars at the single potential N-glycosylation site. Mass spectrometric analysis revealed that the receptor was palmitoylated at the two potential cysteines in its cytoplasmic domain. In conclusion, the full-length CD-MPR produced in P. pastoris is structurally and functionally suitable for crystallization studies.     

Recombinant goldfish growth hormones (gfGH-I and -II) expressed in Escherichia coli have similar biological activities

Complementary DNA regions coding for two different mature goldfish growth hormones (gfGH-I and gfGH-II) with four and five cysteine residues were cloned into the bacterial expression vector, pRSETA. The recombinant gfGH-I (five cysteines) and -II (four cysteines) were produced in Escherichia coli as the fusion proteins carrying N-terminal 6XHis tag, which facilitates purification by using metal chelating affinity chromatography under denaturing condition with urea. The recombinant hormones were further refolded by gradually removing the urea. Native gfGH was also purified from goldfish pituitary glands and served as a positive control in the present study. The native and recombinant hormones were tested in goldfish hepatic radioreceptor assay and in vitro Spi 2.1 promoter activation assay. Our results showed that the two recombinant gfGHs are biologically active, and they have similar biological activities despite their having different cysteine contents.     

Cloning and expression of zebrafish neuronal nicotinic acetylcholine receptors

We propose to use the zebrafish (Danio rerio) as a vertebrate model to study the role of neuronal nicotinic acetylcholine receptors (nAChR) in development. As a first step toward using zebrafish as a model, we cloned three zebrafish cDNAs with a high degree of sequence similarity to nAChR beta3, alpha2 and alpha7 subunits expressed in other species. RT-PCR was used to show that the beta3 and alpha2 subunit RNAs were present in zebrafish embryos only 2-5hours post-fertilization (hpf) while alpha7 subunit RNA was not detected until 8hpf, supporting the differential regulation of nAChRs during development. In situ hybridization was used to localize zebrafish beta3, alpha2, and alpha7 RNA expression. nAChR binding techniques were used to detect the early expression of two high-affinity [3H]-epibatidine binding sites in 2 days post-fertilization (dpf) zebrafish embryos with IC(50) values of 28.6pM and 29.7nM and in 5dpf embryos with IC(50) values of 28.4pM and 8.9nM. These studies are consistent with the involvement of neuronal nAChRs in early zebrafish development.