The manner in which neuronal cells can distinguish the activation of second messenger systems by several receptor types is unclear. We surmise that instead of distinguishing the differences in the magnitude of activation or compartmentation of the second messengers, neuronal cells could distinguish the activation of a specific receptor by integrating the network of multiple effector systems which are simultaneously or sequentially activated by the occupancy of the receptor. If this is the case, then a single receptor type which transduces its signal via the family of G- proteins will interact with multiple G-proteins. We will test this hypothesis by investigating the identities of the G-proteins which can interact with delta-opioid receptor in neuroblastoma x glioma NG108-15 cells, and those which can interact with mu- and delta-opioid receptor in human neuroblastoma SH-SY-5Y. The identification of these G-proteins and the subsequent effectors which can be regulated by these G-proteins will also enable us to better understand the cellular adaptational processes during chronic agonist treatment. The identities of the G-proteins in these two clonal cell lines which can interact with the opioid receptors will be determined initially by utilizing the ability of cholera toxin (CTX) to catalyse the ADP- ribosylation of the G-proteins other than G8 or transducins when they are coupled to the receptor. Therefore, CTX catalysed ADP-ribosylation with 32P-NAD+ will be carried out in the presence of various concentrations of selective opioid agonists and antagonists. Different G-proteins will be separated by urea gradient/SDS PAGE and their initial identities determined by aligning the proteins which are being ADP-ribosylated with the positive bands obtained from western analysis of the blots using specific Galpha antisera. The ability of various treatment which have been reported to alter opioid receptor function, e.g. pertussis toxin treatment, chronic agonist treatment or differentiation, to alter the CTX catalysed ADP- ribosylation will be determined. Because of the homology among various Galphas, the final identities of the G-proteins will be demonstrated by isolation of the Galphas, sequence analysis of the proteolytic fragments and the nucleotide sequence of the isolated cDNA clones. From the deduced peptide sequences, specific polyclonal antibodies to the Galphas will be developed. The ability of these antibodies to modulate the cellular responses to opioid receptor will be determined. This will be carried out by first measuring the alteration in the second messengers level in single cells in the presence of opioid agonists. Affinity purified antibodies of the specific G-proteins will be introduced into the cells and the ability of the antibodies to attenuate the agonist responses will be measured. The G-proteins and their subsequent effectors which can be activated by the delta-opioid receptor will be compared with those which can be activated by mu-opioid receptor. This spectrum of G-proteins will also be compared with that of muscarinic and alpha2-adrenergic receptors. The question of how these two neuronal cell lines could distinguish the activation of these receptors will then be addressed.
Showing the most recent 10 out of 71 publications
