guanylyl-imidodiphosphate and Chromosome-Deletion

The beta-adrenergic receptor (beta AR) contains significant amounts of N-linked carbohydrate. Deletion mutants spanning the four consensus glycosylation sites on the receptor and single amino acid substitutions within the two amino-terminal consensus glycosylation sites reveal that both the amino-terminal sites are utilized. None of the glycosylation-defective beta AR mutants exhibited altered ligand binding in transient expression assays. In addition, the mutant beta ARs which were completely devoid of carbohydrate were capable of coupling to Gs and stimulating adenylyl cyclase in stable L cell lines. In contrast to the wild-type beta AR, the glycosylation-deficient beta ARs expressed in these cells showed a 50% decrease in the level of accumulation on the cell surface. Therefore, while glycosylation of the beta AR does not appear to be essential for receptor function, it is important for correct trafficking of the beta AR protein through the cell.

Topics: Adenylyl Cyclases; Animals; Base Sequence; Cell Line; Chromosome Deletion; Cloning, Molecular; Cricetinae; DNA; Glucosamine; Glycosylation; Guanylyl Imidodiphosphate; Isoproterenol; Molecular Sequence Data; Mutation; Oligonucleotide Probes; Receptors, Adrenergic, beta; Transfection; Tunicamycin

The adenylate cyclase system of the yeast Saccharomyces cerevisiae contains the CYR1 polypeptide, responsible for catalyzing formation of cyclic AMP (cAMP) from ATP, and two RAS polypeptides, which mediate stimulation of cAMP synthesis of guanine nucleotides. By analogy to the mammalian enzyme, models of yeast adenylate cyclase have depicted the enzyme as a membrane protein. We have concluded that adenylate cyclase is only peripherally bound to the yeast membrane, based on the following criteria: (i) substantial activity was found in cytoplasmic fractions; (ii) activity was released from membranes by the addition of 0.5 M NaCl; (iii) in the presence of 0.5 M NaCl, activity in detergent extracts had hydrodynamic properties identical to those of cytosolic or NaCl-extracted enzyme; (iv) antibodies to yeast adenylate cyclase identified a full-length adenylate cyclase in both membrane and cytosol fractions; and (v) activity from both cytosolic fractions and NaCl extracts could be functionally reconstituted into membranes lacking adenylate cyclase activity. The binding of adenylate cyclase to the membrane may have regulatory significance; the fraction of activity associated with the membrane increased as cultures approached stationary phase. In addition, binding of adenylate cyclase to membranes appeared to be inhibited by cAMP. These results indicate the existence of a protein anchoring adenylate cyclase to the membrane. The identity of this protein remains unknown.

Topics: Adenylyl Cyclases; Cell Membrane; Chromatography, Gel; Chromosome Deletion; Cytoplasm; Gene Expression; Genotype; Guanosine Diphosphate; Guanylyl Imidodiphosphate; Kinetics; Membrane Proteins; Methionine; Molecular Weight; Plasmids; Saccharomyces cerevisiae; Sulfur Radioisotopes; Thionucleotides

The molecular basis for the desensitization of beta 2-adrenergic receptors was investigated by oligonucleotide-directed mutagenesis. beta-Adrenergic receptor mutants containing deletions within the sixth hydrophilic domain that failed to couple to Gs and stimulate adenylyl cyclase did not undergo agonist-mediated sequestration. In contrast, all receptor mutants that displayed Gs coupling were sequestered away from the cell surface in response to isoproterenol. Progressive truncation of the C-terminus of the receptor resulted in decreases in the initial rates of receptor sequestration and functional uncoupling, although the final extent of these desensitization processes was not affected by the mutations. These data suggest that structural features of the beta 2-adrenergic receptor that are involved in receptor activation are also essential for mediating the subsequent inactivation caused by the sequestration of the receptor from the cell surface.

Topics: Adenylyl Cyclases; Adrenergic beta-Agonists; Animals; Chromosome Deletion; Cricetinae; GTP-Binding Proteins; Guanylyl Imidodiphosphate; Isoproterenol; Mutation; Protein Conformation; Receptors, Adrenergic, beta