Activation of muscarinic cholinergic recoptors (MR) results in both an inhibition of adenylate cyclase (AC) and a breakdown of phosphoinositides (PI). Our goal is to define the molecular basis of these biochemical responses. Two cell lines will be utilized as model tissues for the study of MR that couple to AC (NG109-15 neuroblastoma x glioma cells) or stimulate the breakdown of P1 (1321N1 astrocytoma cells). Recent work suggests that separate MR subtypes exist on these two cells. Also, indirect evidence is consistent with the idea that different guanine nucleotide regulatory proteins (N-proteins) are involved in transducing the two biochemical responses mediated by the two putative MR subtypes. The research is designed to confirm or refute the idea that specific MR subtypes exist that couple to two different N-proteins to produce two different biochemical responses. Plasma membrane preparations from NG108-15 and 1321N1 cells will be solubilized and MR and N-protein activities resolved by chromatographic techniques. The resolved MR will be reconstituted into model phospholipid vesicles and standard radioligand binding assays carried out to determine if the reconstituted MR retain their known pharmacological specificities. MR then will be reconstituted with resolved N-protein fractions with the goal of reestablishing negative heterotropic effects of guanine nucleotides on agonist binding to MR. NG108-15 and 1321N1 cell preparations will be treated with toxins and alkylating agents to differentially inactivate the various N-proteins known or proposed to be present in these cells. These treated preparations will be reconstituted with MR to determine the selectivity of interaction of the putative MR subtypes with N-proteins. One goal of these experiments will be to determine if a previously undescribed N-protein is present that couples to the putative MR subtype that stimulates P1 breakdown. This question will be addressed further by reconstituting purified N-proteins (Ns, Ni, N0, transducin, and Np, a protein purified from placenta) with the MR from the two cell lines. MR-N-protein interaction again will be assessed by observation of regative heterotropic effects of guanine nucleotides on agonist binding to MR. Successful reconstitution of each putative MR subtype with one or more N-proteins will be further characterized by measuring the properties of MR-stimulated 35S-GTP Gamma S binding and GTPase activity in the vesicle preparations.
| Liang, M; Martin, M W; Harden, T K (1987) [3H]propylbenzilylcholine mustard-labeling of muscarinic cholinergic receptors that selectively couple to phospholipase C or adenylate cyclase in two cultured cell lines. Mol Pharmacol 32:443-9 |
