RECEPTOR-MEDIATED INHIBITION OF ADENYLATE CYCLASE

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The long term objective of this research plan is to gain insight into the mechanism of action of receptors inhibiting adenylate cyclase activity. Numerous neuronal receptors including opiate, D-2 dopaminergic, alpha2-adrenergic, purinergic, and cholinergic are believed to modulate neuronal activity by inhibiting adenylate cyclase. Since these receptors play a critical role in modulating the functioning of the brain, insight into their mechanism of action is fundamental to the understanding and treatment of neurological disorders involving these receptors. Recently, a Mu-opiate receptor with pharmacological properties similar to brain Mu-opiate receptors was discovered on the cells of a prolactin secreting tumor termed 7315c. Preliminary results suggest that this receptor is associated with the inhibitory GTP binding protein, termed Ni, which acts as a transducer between the inhibitory receptor and adenylate cyclase. The specific aim of this research proposal is to determine how the Mu-opiate receptor enhances the interaction of guanyl nucleotides with Ni. It is hypothesized that activation of the Mu-opiate receptor enhances the exchange of GDP for GTP at Ni. Initially, opiates will be tested for their ability to stimulate GTPase activity in 7315c membranes. Next, GDP will be tested for its ability to block both the Gpp(NH)p- and the GTP-induced activation of Ni. Opiate agonists will also be tested for their ability to enhance the removal of Gpp(NH)p from Ni and cause predictable changes in adenylate cyclase activity. Pertussis toxin has recently been proposed to induce an ADP ribosylation of Ni resulting in the uncoupling of Ni and inhibitory receptors. Pertussis toxin will be tested for its ability to block Mu-opiate receptor-mediated exchange of guanyl nucleotides at Ni. It will also be determined if pertussis toxin has a direct effect on inhibitory receptors. Intermediate lobe membranes containing an inhibitory D-2 dopamine receptor will be treated with pertussis toxin and then fused with 7315c membranes (which contain no D-2 receptor). The D-2 dopamine receptor will then be tested for its ability to recouple with Ni and inhibit adenylate cyclase. Finally, an opiate affinity column will be used in an attempt to isolate the Mu-opiate receptor in close association with Ni.