Evidence for the involvement of adenomatous polyposis coli (APC) protein in maintaining cellular distributions of α3ß4 nicotinic receptors.

ABSTRACT

Evidence exists supporting the involvement of adenomatous polyposis coli (APC) protein in the assembly of neuronal nicotinic acetylcholine receptors (nAChRs) in the postsynaptic complex. In the following studies, the effects of APC protein on cellular distribution of recombinant α3ß4 nAChRs was investigated. RT-PCR and Western blotting techniques established the expression of APC protein both in bovine adrenal chromaffin cells, which express native α3ß4* nAChRs, and in a HEK293 cell line expressing recombinant bovine adrenal α3ß4 nAChRs (BMα3ß4 cells). Transfection of BMα3ß4 cells with siRNA to APC, reduced APC protein levels to 52.4% and 61.9% of control values at 24 and 48 h after transfection. To investigate the effects of APC on the cellular distribution of α3ß4 nAChRs, [(3)H]epibatidine binding approaches, coupled with APC siRNA treatment, were used. Twenty-four and 48 h after APC siRNA transfection, intracellular nAChRs were significantly reduced to 71% and 68% of control, respectively, while the total population of nAChRs were not significantly changed. Given that total cellular nAChRs represent the sum of surface and intracellular nAChRs, these studies support a re-distribution of nAChRs to the plasma membrane with APC siRNA treatment. Treatment of the cells with the protein synthesis inhibitor, puromycin, also caused a significant reduction (55%) in APC protein levels, and produced a similar re-distribution of cellular nAChRs. These studies support the involvement of APC protein in the maintenance of normal cellular distribution of α3ß4 nAChRs.