Quantification of cell surface Mu and Delta opioid receptor and elucidation of factors governing surface receptor pool.

Abstract

We developed a method of quantifying cell surface and total cell receptor in individual cells by separately labeling these populations of receptor with different fluorescent antibodies, acquiring images with fluorescence microscopy, and quantifying fluorescence for each cell using Image J software. Using this method, we studied the surface-to-total ratios for transfected mu opioid receptor (MOR) and delta opioid receptor (DOR) in PC12 cells and HEK cells. We found that the average surface-to-total ratio was higher for DOR than for MOR in both cell types, and regardless of NGF differentiation of the PC12 cells, indicating the existence of receptor-specific installation mechanisms for these receptors. For both receptors, we found little relationship between the quantities of cell surface and total cell receptor, but surface-to-total ratio decreases at high levels of total receptor, suggesting that the mechanisms of receptor installation are saturable. In NGF-differentiated PC12 cells, we found that either depolarization using potassium chloride or co-expression of the Substance P precursor pre-pro-tachykinin increased the surface-to-total ratio of DOR but not MOR, consistent with a highly specific role for the regulated secretory pathway in cell surface delivery of DOR. We found that the DOR agonist deltorphin could depress DOR surface-to-total ratio for significantly longer than would be expected based on normal trafficking kinetics, showing that agonist treatment can cause receptor down-regulation not only by direct internalization but also by producing a longer-lasting change in receptor membrane trafficking. Finally, we found that in PC12 cells co-expressing DOR and MOR, treatment with the DOR agonist deltorphin for 30 minutes caused a rapid, significant increase in cell surface MOR, but treatment with the MOR agonist DAMGO for the same time period did not cause an increase in cell surface DOR. These experiments point to a multitude of receptor-specific factors that determine the quantity of DOR and MOR on the cell surface.