guanylyl-imidodiphosphate and Adrenal-Cortex-Neoplasms

Mutant isolates [designated desensitization resistant (DR)] from the Y1 mouse adrenocortical tumor cell line resist agonist-induced desensitization of adenylyl cyclase by preventing the uncoupling of receptors from their guanyl nucleotide-binding regulatory G proteins. In this study, we tested the hypothesis that an underlying G protein defect is associated with the DR phenotype. We found that the G protein reagent guanyl-5'-yl imidodiphosphate [Gpp(NH)p] shifted beta2-adrenergic receptors from a high affinity state to a low affinity state 4-fold more effectively in mutant DR cells than in parent Y1 cells. In the DR mutant, Gpp(NH)p was able to shift receptors to a low affinity state in the absence of NaCl, whereas the effect of Gpp(NH)p in parent Y1 cells was dependent upon the presence of NaCl. Moreover, these differences in sensitivity to Gpp(NH)p and NaCl were transferred to Gs alpha-deficient S49(CYC-) lymphoma cell membranes in G protein reconstitution assays. These observations suggested that the DR mutation was associated with altered activity of the stimulatory G protein, Gs. Cloning and sequence analysis demonstrated that Gs alpha transcripts in the DR mutant were normal, suggesting that another factor involved in guanyl nucleotide exchange is responsible for the altered G protein activity in DR mutant cells.

Topics: Adaptation, Physiological; Adrenal Cortex Neoplasms; Amino Acid Sequence; Animals; Base Sequence; DNA, Complementary; GTP-Binding Proteins; Guanylyl Imidodiphosphate; Lymphoma; Mice; Molecular Sequence Data; Mutation; Phenotype; Receptors, Adrenergic, beta; Sodium Chloride; Tumor Cells, Cultured

The properties of the adenylate cyclase from forskolin-resistant mutants of Y1 adrenocortical tumor cells was compared with the properties of the enzyme from parental Y1 cells in order to localize the site of mutation. In parental Y1 cells, forskolin stimulated adenylate cyclase activity with kinetics suggestive of an interaction at two sites; in mutant cells, forskolin resistance was characterized by a decrease in enzymatic activity at both sites. Forskolin potentiated the enzyme's responses to NaF and guanyl-5'-yl imidodiphosphate (Gpp(NH)p) in parent and mutant clones, and the mutant enzyme showed the same requirements for Mg2+ and Mn2+ as did the parent enzyme. The adenylate cyclase associated with forskolin-resistant mutants was insensitive to ACTH and was less responsive to Gpp(NH)p than was the parent enzyme. In parental Y1 cells and in the forskolin-resistant mutants, cholera toxin catalyzed the transfer of [32P]ADP-ribose from [32P]NAD+ into three membrane proteins associated with the alpha subunit of Gs; however, the amount of labeled ADP-ribose incorporated into mutant membranes was reduced by as much as 70%. Both parent and mutant membranes were labeled by pertussis toxin to the same extent. The insensitivity of the mutant adenylate cyclase to ACTH and Gpp(NH)p and the selective resistance of the mutant membranes to cholera toxin-catalyzed ADP-ribosylation suggest that a specific defect associated with Gs is involved in the mutation to forskolin resistance in Y1 cells.

Topics: Adenosine Diphosphate Ribose; Adenylyl Cyclases; Adrenal Cortex Neoplasms; Adrenocorticotropic Hormone; Animals; Colforsin; Drug Resistance; GTP-Binding Proteins; Guanylyl Imidodiphosphate; Kinetics; Magnesium; Manganese; Mice; Mutation; NAD; Phenotype; Sodium Fluoride

The present investigation was performed in order to study the properties of abnormal membrane function related to ACTH receptor-adenylate cyclase system interaction in human ACTH-unresponsive adrenocortical cancer. Two tissues of adrenocortical cancer obtained from a patient with Cushing's syndrome (CS) and a case presenting no abnormal endocrinological findings (NF) were used for in vitro studies, comparing with three normal adrenal tissues. The addition of ACTH alone and ACTH plus 10(-6) M GppNHp did not enhance the adenylate cyclase (AC) activity in the CS and NF tissues. Relative insensitivity of AC to GTP, GppNHp, and cholera toxin was observed for the NF tissue, while the rate of response to GppNHp for the CS tissue which also showed relative insensitivity to GTP and cholera toxin was similar to that for the normal tissues. Forskolin which is reported to directly activate the catalytic unit of the AC complex increased the AC activity of both CS and NF tissues as well as that of the normal tissues. Therefore, the function of the catalytic unit itself may be rather well preserved in these tumor tissues. These results suggest that the lack of ACTH receptor at the cell membrane surface might be responsible for ACTH-unresponsiveness in the CS tissue, although an accelerated degradation of GTP could contribute to decreased activity of GTP-binding protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenylyl Cyclases; Adrenal Cortex Neoplasms; Adrenocorticotropic Hormone; Carcinoma; Cell Membrane; Cholera Toxin; Colforsin; GTP-Binding Proteins; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Humans; Receptors, Corticotropin; Receptors, Pituitary Hormone

Fractions enriched in plasma membranes were prepared from the Y1 mouse adrenocortical tumor cell line and were characterized with respect to adenylate cyclase activity. Optimal requirements of the adenylate cyclase system for guanyl nucleotides. Mg2+, ATP, and corticotropin (ACTH) were determined. The sensitivity of the adenylate cyclase system to ACTH in plasma membrane fractions was comparable with that observed in isolated intact cells. Polycations such as poly-L-arginine and histone competitively inhibited the action of ACTH, supporting the view that the affinity of ACTH for the adenylate cyclase system is determined by the basic core of amino acids at residues 15-18. ACTH was at least one order of magnitude more potent than ACTH in stimulating adenylate cyclase activity in plasma membrane fractions.

Topics: Adenosine Triphosphate; Adenylyl Cyclases; Adrenal Cortex Neoplasms; Adrenocorticotropic Hormone; Animals; Cell Line; Cell Membrane; Guanylyl Imidodiphosphate; Magnesium; Magnesium Chloride; Mice

Adrenocorticotropin(ACTH)-induced desensitization of adenylate cyclase was examined in subclones derived from the ACTH-responsive, Y1 mouse adrenocortical tumor cell line. This report describes clonal variation in ACTH-induced desensitization of adenylate cyclase and an associated variation in the level of a 68,000-dalton protein, p68. A subclone of Y1 cells with a low level of p68 (0.8% of total protein) exhibited a faster rate of desensitization and a slower rate of recovery from desensitization when compared with a clone containing a high level of p68 (10% of total protein). In three clones with low levels of p68, ACTH desensitized adenylate cyclase with ED50 values from 0.3 to 0.5 nM. In several clones with high levels of p68, the adenylate cyclase system was more resistant to ACTH-induced desensitization; the ED50 values for ACTH in these clones ranged from 2 to 12 nM. Among 11 ACTH-responsive subclones, the level of p68 correlated significantly (p less than 0.001, r = 0.87) with resistance to the desensitization induced by 1 nM ACTH. These results suggest that p68 may function in the maintenance of an ACTH-responsive adenylate cyclase system, or that the level of p68 and responsiveness to ACTH are coordinately regulated.

Topics: Adenylyl Cyclases; Adrenal Cortex Neoplasms; Adrenocorticotropic Hormone; Animals; Cell Line; Clone Cells; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Kinetics; Mice; Molecular Weight; Neoplasms, Experimental

Topics: Adrenal Cortex Neoplasms; Adrenocorticotropic Hormone; Animals; Calcium; Enzyme Activation; Fluorides; Guanylate Cyclase; Guanylyl Imidodiphosphate; Neoplasms, Experimental; Solubility; Subcellular Fractions

NPS(o-nitrophenyl sulfenyl)-ACTH stimulated steroidogenesis in Y1 cells to the same maximum level as did ACTH, but with a 60-fold lower potency than the native hormone. NPS-ACTH also stimulated the extracellular accumulation of cAMP in Y1 cells with lower potency than the unmodified hormone. The amount of cAMP accumulated in the presence of NPS-ACTH approached 75% of the maximum with ACTH. In Y1 plasma membranes, NPS-ACTH was a partial agonist of adenylate cyclase activity, witn an efficacy dependent upon the type of guanyl nucleotide present. The steroidogenic responses of two Y1(Kin) mutants and two Y1(Cyc) mutants to NPS-ACTH paralleled their responses to ACTH and reflected closely their defects in cAMP-dependent protein kinase and ACTH-sensitive adenylate cyclase activity. These data imply an obligatory role for adenylate cyclase and cAMP-dependent protein kinase activities in stimulation of steroidogenesis in Y1 cells by NPA-ACTH.

Topics: Adenylyl Cyclases; Adrenal Cortex Neoplasms; Adrenocorticotropic Hormone; Animals; Clone Cells; Cyclic AMP; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Mice; Neoplasms, Experimental; Protein Kinases; Steroids

Topics: Adenylyl Cyclases; Adrenal Cortex Neoplasms; Adrenal Gland Neoplasms; Adrenocorticotropic Hormone; Diphosphonates; Drug Resistance; Female; Guanine Nucleotides; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Humans; Hydrocortisone; In Vitro Techniques; Prostaglandins E; Receptors, Drug; Receptors, Prostaglandin