Receptor-G-protein coupling in desensitization-resistant mutant adrenal cells

Mutant isolates from the Y1 mouse adrenocortical tumor cell line (designated Y1DR) were previously shown to resist homologous desensitization of adenylyl cyclase via endogenous ACTH receptors or transfected beta 2-adrenergic receptors. To further localize the site of the DR mutation, we examined the agonist-binding properties of the transfected beta 2-adrenergic receptor in parental Y1 cells and the DR mutant before and after desensitization with the beta-adrenergic agonist isoproterenol. Binding studies were carried out using [125I]iodocyanopindolol as the labeled ligand and isoproterenol as the competing agonist. We found that the DR mutant has a greater number of high affinity receptors in the basal state than does the Y1 parent (70-80% vs. < or = 50%) and that treatment of membranes from parent or mutant cells with guanyl-5'-yl imidodiphosphate and NaCl converts all of the high affinity sites to low affinity sites. After desensitization with isoproterenol, only low affinity binding sites are detected in parental Y1 cells, whereas the DR mutants retain an appreciable number of high affinity receptors. These results indicate that the DR mutant may resist desensitization by affecting agonist-induced uncoupling of receptors from their guanyl nucleotide-binding regulatory G-proteins

Heterologous desensitization of the human dopamine D1 receptor in Y1 adrenal cells and in a desensitization-resistant Y1 mutant

In previous studies, mutant clones (designated Y1DR) were isolated that resisted ACTH-induced homologous desensitization of adenylyl cyclase. The Y1DR mutation also conferred resistance to the homologous desensitization induced by agonist stimulation of transfected beta 2-adrenergic receptors. These observations suggested that ACTH and beta 2-adrenergic agonists homologously desensitized adenylyl cyclase in Y1 cells by a common mechanism. In the present study, parental Y1 cells (Y1DS) and the Y1DR mutant were transfected with the gene encoding the human dopamine D1 receptor and examined for regulation of adenylyl cyclase by dopaminergic agonists. Transformants were isolated from both cell lines and shown to respond to dopamine agonists with increases in adenylyl cyclase activity. Treatment of the Y1DS transformants with ACTH promoted a rapid, homologous desensitization of adenylyl cyclase and had little effect on the responses to dopamine or NaF; treatment of Y1DS with dopaminergic agonists promoted a slower rate of heterologous desensitization that diminished responsiveness of the adenylyl cyclase system to dopamine, ACTH, and NaF. Y1DR cells transfected with the dopamine D1 receptor were resistant to the heterologous desensitization of adenylyl cyclase induced by dopaminergic agonists. These latter observations suggest that the pathways of homologous desensitization and heterologous desensitization converge at a common point in the desensitization pathway defined by the DR mutation in Y1 cells

Molecular basis for the 3',5'-cyclic adenosine monophosphate resistance of Kin mutant Y1 adrenocortical tumor cells

A series of mutant cell lines (Kin) were previously isolated from Y1 adrenocortical tumor cells based on their ability to resist the growth-inhibitory effects of 8-bromo cAMP. In these Kin clones, cAMP-dependent protein kinase (cAMPdPK) was resistant to activation by cAMP as the consequence of mutations affecting the type I regulatory subunit (RI) of the enzyme. This study shows that the cAMP-resistant phenotypes of mutant clones Kin-2, Kin-7, and Kin-8 were associated with single base changes causing substitutions, respectively, of Glu for Gly200, Trp for Arg334, and Asp for Gly324 in the RI protein. By expressing the mutant Trp334 and Asp324 forms of RI under the control of an inducible promoter in Y1 cells, the causal relationship between these RI mutations and impairment of cAMP-stimulated adrenocortical responses was studied. Expression of the mutant RI forms rendered cAMPdPK resistant to activation by cAMP and decreased cAMP-stimulated cell rounding, steroid production, and growth inhibition. These observations indicate that the cAMP-resistant phenotype of Kin mutant clones resulted specifically from single mutational events in RI and thus establish the importance of cAMPdPK as an essential regulator of adrenocortical function. Unlike the original Kin mutant clones, transformants expressing the mutant forms of RI had adenylyl cyclases that were resistant to activation by ACTH, forskolin, or sodium fluoride. These results indicate that there may be a hitherto unappreciated mechanism of regulation of adenylyl cyclase activity by cAMPdPK

A New Symbolic Representation for the Identification of Informative Genes in Replicated Microarray Experiments

Microarray experiments generate massive amounts of data, necessitating innovative algorithms to distinguish biologically relevant information from noise. Because the variability of gene expression data is an important factor in determining which genes are differentially expressed, analysis techniques that take into account repeated measurements are critically important. Additionally, the selection of informative genes is typically done by searching for the individual genes that vary the most across conditions. Yet because genes tend to act in groups rather than individually, it may be possible to glean more information from the data by searching specifically for concerted behavior in a set of genes. Applying a symbolic transformation to the gene expression data allows the detection overrepresented patterns in the data, in contrast to looking only for genes that exhibit maximal differential expression. These challenges are approached by introducing an algorithm based on a new symbolic representation that searches for concerted gene expression patterns; furthermore, the symbolic representation takes into account the variance in multiple replicates and can be applied to long time series data. The proposed algorithm's ability to discover biologically relevant signals in gene expression data is exhibited by applying it to three datasets that measure gene expression in the rat liver