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Relaxation of the trabecular smooth muscle, which is necessary for penile erection, is controlled locally by neurotransmitters and vasoactive agents. The goal of this study was to identify and characterize muscarinic acetylcholine receptor (mAChR) subtypes expressed in cultured human corpus cavernosum smooth muscle cells (HCC SMC). Binding analysis with L-[benzilic-4,4'-3H(N)]quinuclidinyl benzilate ([3H]QNB) demonstrated the expression of specific muscarinic receptor binding sites in HCC SMC. Analysis of total RNA isolated from whole corpus cavernosum tissue and smooth muscle cells, by RNase protection assays, demonstrated the expression of mRNA transcripts for m1, m2, m3, and m4 mAChR subtypes in whole tissue and m2 and m4 subtypes in cultured cells. In situ hybridization with specific m2 and m4 probes further confirmed the expression of m2 and m4 mRNA transcripts in cultured cells. Carbachol (CCh), a nonselective cholinergic agonist, inhibited cAMP synthesis at low concentrations (0.1-1 microM) and stimulated cAMP synthesis at high concentrations (100 microM), in cultured HCC SMC. CCh (100 microM) further augmented forskolin (FSK), isoproterenol (ISO), and prostaglandin E1 (PGE1)-induced cAMP synthesis. These observations suggest that, in vivo, in HCC, ACh may activate m3 mAChR subtypes on endothelial cells or m2 and m4 subtypes on the SMC. Although m2 and m4 are thought to inhibit adenylate cyclase (AC), the augmentation of cAMP synthesis by high concentrations of CCh in SMC suggests an alternative mechanism of coupling to G-proteins that stimulates AC activity. These studies show that HCC tissue expresses different subtypes of mAChR (m1, m2, m3, and m4), whereas cultured HCC SMC express m2 and m4 subtypes. It is suggested that m2 and m4 receptor subtypes may play an important role in maintaining trabecular smooth muscle tone in vivo. The augmentation of FSK-, ISO, and PGE1-induced cAMP synthesis by CCh suggests possible development of a multidrug therapeutic approach to treatment of erectile dysfunction.

Studies of naturally occurring and chemically modified insulins indicate that relatively few of the 51 amino acid residues may be assigned specific roles in insulin-receptor interactions. Most of the insulin X-ray structural information is derived from aggregated species (notably hexamers). Because insulin exerts its physiological effect as a 5808 Dalton monomeric species, it is necessary to consider whether crystal-packing forces have modified the structure from that required for biological action. Insulin aggregation in solution complicates high resolution NMR studies of the monomer. However, site-directed mutagenesis can be used to generate biologically active mutants (e.g., B16-Tyr--> His) that remain monomeric at millimolar concentrations in aqueous solution at low pH. The resulting homogeneous and monomeric samples are suitable for structure determination by NMR methods. The high resolution solution structure of B16--Tyr--> His insulin resembles crystal structures, notably molecule 1 of T6 insulin. Side-chain conformation in some biologically important motifs, however, shows subtle differences between solution and crystal structures.

Cyclic ADP-ribose is a recently discovered metabolite of NAD that appears to function in cellular calcium signaling. The discovery that NAD glycohydrolases are bifunctional enzymes that catalyze both the synthesis and hydrolysis of cyclic ADP-ribose raises many questions concerning the mechanisms by which these enzymes function in calcium signaling. Likewise, the identification of human lymphocyte antigen CD 38 as a bifunctional NAD glycohydrolase raises interesting questions concerning the involvement of cyclic ADP-ribose mediated calcium signaling in immune function. The dementia associated with niacin deficiency has been a long-standing curiosity. This signaling mechanism may resolve questions connecting this vitamin deficiency to central nervous system (CNS) function.

Following the growth hormone (GH) and GH receptor (R) interaction, the receptor and Janus tyrosine kinase 2 (JAK2) become tyrosine phosphorylated along with other intracellular proteins. Previously, we reported that GH induces tyrosine phosphorylation of intracellular proteins with molecular masses of approx 95 kDa (pp95) in mouse 3T3-F442A preadipocytes and in mouse L-cells that express recombinant GHRs. We have studied this GH-induced phosphorylation event in greater detail. Three proteins with apparent molecular masses of 93, 95, and 96 kDa showed increased tyrosine phosphorylation in a time-dependent manner following GH treatment of cells that express GH receptors. GH-induced tyrosine phosphorylation of these proteins is independent of activation of protein kinase C (PKC). Cell fractionation studies revealed that the majority of tyrosine-phosphorylated pp95/96 is located in the cytoplasm. pp95 and pp96 have pIs of approx 6.2. Immunoprecipitation and Western blot analyses revealed that pp93 and pp95/96 are not immunologically related with Stat1, Stat3, Stat4, JAK2, and GHR. Thus, pp93 and pp95/96 may be important GH signal transducers independent of PKC activation and different from the characterized members in the JAK-STAT pathway.

Glucocorticoids stimulate surfactant protein-B (SP-B) (expression in type II alveolar cells) by unknown mechanisms. We identified, cloned, and characterized a protein that binds the SP-B promoter. This protein, D, increases the activity of the SP-B promoter in response to glucocorticoid stimulation. Protein D was identified by its ability to bind the SP-B promoter region, which it binds at an NF1 site from -184 to -198 bp. Its binding was abolished by digestion of promoter DNA with BalI, which cuts at -194. Protein D was cloned and sequenced. It is a new DNA binding protein of 33 kDa whose carboxyl end contains a modified basic leucine zipper-like DNA binding motif (bzip). The effects of D on SP-B promoter activity were studied in H441 cells, using a reporter construct containing 212 bp from the SP-B promoter with a luciferase reporter gene (p2121uc), which was cotransfected with a protein D expression construct in which D expression was controlled by the SV40 early promoter. These two plasmids were cotransfected into H441 cells. Without added glucocorticoids, D did not alter SP-B promoter activity. When dexamethasone was added, D strongly enhanced SP-B promoter activity. Identification of this protein suggests that, at least for SP-B, glucocorticoid responsiveness may involve one or more hitherto unknown gene activators.

We studied the effects of changing beta 1- and beta 2-adrenergic receptor (AR) subtype ratios and densities on cyclic AMP (cAMP) responses to norepinephrine (NE) and epinephrine (EPI) in rat C6 glioma cells. Dexamethasone (DEX) increased beta 2- and decreased beta 1-AR expression without changing total beta-AR density, whereas pretreatment with selective agonists specifically downregulated each subtype. Combinations of these treatments produced cells with six different beta 2/beta 1 ratios that ranged from 0 (100% beta 1) to 2.85. We compared the effects of NE and EPI on cAMP accumulation in each condition and observed a predominantly beta 1 pharmacology (NE > EPI) under most conditions. However, as the beta 2-AR density exceeded the number of beta 1-ARs we observed a progressive shift toward a more beta 2-like pharmacology (EPI > NE), without the appearance of biphasic concentration-response curves. The ratio of beta 2/beta 1 density correlated significantly (p < 0.006) with the ratio of the potencies of NE and EPI in increasing cAMP formation. We conclude that in native C6 cells beta 1-ARs appear to couple more efficiently to cAMP accumulation than do beta 2-ARs, but both subtypes contribute to catecholamine responses in a nonadditive manner when the proportion of beta 2-ARs is increased.

The low-calorie nutrient-dense diet consumed for 2 yr by the eight persons sealed inside the closed ecological space known as Biosphere 2, near Tucson, AZ, constituted a unique "experiment of nature," amounting to the first well-monitored application of a nutritional regimen proven in animals to substantially inhibit and delay time of onset of most age-related diseases, induce physiological changes characteristic of a functionally "younger" age, and extend both average and maximum lifespans. Over the 2 yr the eight persons demonstrated a substantial weight loss, remarkable fall in blood cholesterol, blood pressure, fasting blood sugar, and low white blood cell counts--exactly as seen in rodents on such a regimen. Studies in progress involving levels of cortisol, insulin, and glycosylated hemoglobin support the rodent similarity. Further studies will seek to determine whether additional among the large battery of physiologic changes induced in animals by caloric restriction are also induced in humans on a similar nutrient-dense, calorie-limited diet. Such evidence will pertain to the question whether the increased disease resistance and aging retardation shown by calorie-restricted rodents might also be expected to occur in humans.

Asparagine-linked glycosylation of the insulin receptor is required for complete biosynthesis and acquisition of function. However, the relative role of each individual glycosylation site has not been elucidated. Previously, it has been shown that removal, by site-directed mutagenesis, of the four amino terminal glycosylation sites (N16,N25,N78, and N111) results in a mutant insulin receptor that remained in the endoplasmic reticulum as an unprocessed proreceptor (Collier E., Carpentier J.-L., Beitz L., Caro L. H. P., Taylor S. I., and Gorden P. [1993] Biochemistry 32, 7818-7823). In the present study, the contribution of these independent glycosylation sites to dimerization and insulin binding has been evaluated. Chinese hamster ovary cells were transfected with the wild-type human insulin receptor cDNA, or cDNA that had Q substituted for N at one, two, or all four of these glycosylation sites. Electrophoretic characterization of the proteins immunoprecipitated from 35S-labeled cells showed that both the wild-type and the quadruple mutant receptor had similar profiles, indicating that the mutant receptor is capable of undergoing dimerization. Analysis of the biochemical properties of this mutant showed that this receptor binds insulin, but ligand binding does not result in kinase stimulation. We demonstrated that the absence of kinase activation is not a property of the mutated receptor since the wild-type proreceptor behaves in a similar manner. Only partial glycosylation in this region of the receptor is required for its targeting to the cell membrane since single and double glycosylation mutants were found processed to their alpha and beta subunits on the cell surface.(ABSTRACT TRUNCATED AT 250 WORDS)

The development of malignant melanoma is accompanied by an accumulation of genetic damage that is evident within tumor cells at both cytogenetic and molecular levels, and mutations at several gene loci are thought to contribute to malignant progression. Some of these loci are known oncogenes and tumor suppressor genes; others remain to be identified, although their chromosomal locations have been determined. Gene mapping studies indicate the presence of melanoma tumor suppressor genes on chromosomes 1, 6, and 9. The presence of a tumor suppressor gene on a particular chromosome can be demonstrated by transfer of an intact, normal copy of the chromosome into tumor cells. We have used this approach to investigate the mechanisms by which chromosome 6 suppresses the growth and tumorigenicity of human malignant melanoma cells.