It remains our overall objective to elucidate the mechanism of how neuronal cells could distinguish activation of multiple G-proteins coupled receptors when they appear to couple to same effector systems. Our hypothesis has been that these receptors could activate multiple G-proteins and cellular distinction lies in ability of neuronal cells to effector systems which are being activated. We tested this hypothesis by investigating delta- opioid receptor coupling to G- proteins in several neuroblastoma cells. By carrying experiments using GTP photoaffinity labels, and the ability of agonist to promote cholera toxin catalysed ADP-ribosylation of cognate arginine moiety in Galphas, we observed that delta-opioid receptor could interact with multiple G-proteins which are pertussis toxin (PTX) substrates in NG108-15, NS20Y and N1E115 cells. To our surprise, the affinity of agonist to promote these interaction is similar for all G-proteins. By taking advantage of different level of expression of cloned receptor in transfected CHO cells, we could demonstrate that receptor- G-protein interaction is a function of receptor density while ability of agonist to inhibit adenylate cyclase is not. These data, together with others, suggested to us that there are cellular proteins other than heterotrimeric G-proteins which are involved in opioid receptor signal transduction. Therefore, in current proposal, we will continue our efforts in identifying the G-proteins involved in opioid receptor signal transduction. We will now examine probable interaction between opioid receptor and G-proteins which are not PTX substrates. We will utilize all the recently cloned opioid receptors, mu-, kappa- and delta-opioid receptor, to establish cell lines in which either homogeneous population of receptor is expressed or a combination of these receptors will be expressed. Cell lines such as AtT20, GH3 and other cells which possess multiple effector systems for probable opioid receptor regulation will be used in these studies. We will also use Xenopus oocytes as expression system for studying opioid receptor-G-protein interactions, using CFTR, a cAMP sensitive chloride channel as a reporter for opioid receptor activity. G-proteins which interact with these receptors will be identified with 32P-alpha- azidoanilido GTP, subsequent separation with urea/SDS PAGE and Galpha- specific antibodies. Effect of one receptor activation on ability of other receptor to interact with G-proteins will be addressed. We will correlate specific G-proteins which are involved in selective opioid receptor actions by use of Galpha specific antisense oligodexoynucleotides so as to eliminate opioid responses and to use Galpha mutated to eliminate PTX-sensitivity so as to retain opioid receptor activity in cells treated with PTX. The opioid receptor responses to be measured will be agonist regulation of intracellular cAMP level, IP3 level, Ca+2 level and overall cell proliferation. The sites on the receptor which interact and might infer G-protein selectivity will be identified by receptor mutagenesis studies. Involvement of other cellular proteins will be identified by receptor mutagenesis studies. Involvement of other cellular proteins will be identified by examining the role of bg-subunits of heterotrimeric G- proteins, smg or GAP-like proteins. The presence of these cellular proteins will be examined in immunoaffinity purified agonist-opioid receptor complexes or complexes generated with constitutively active opioid receptor mutants. The role of these cellular proteins and others in opioid receptor signal transduction will be determined.
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