The interaction of CD4 with the endocytic pathway during down-regulation

CD4 expressed at the surface of lymphoid cells is down-regulated in response to antigenic stimulation. Down-regulation is believed to involve the activation of protein kinase C, and can be mimicked by phorbol esters, such as phorbol myristic acid (PMA), but the cellular mechanisms that result in clearance of CD4 from the cell surface are not understood. In this thesis I describe experiments which analyse phorbol ester-induced down-regulation in detail in HeLa cells that stably express CD4. I show that down-regulation is a multi-step process in which the kinetics and intracellular itinery of CD4 are modulated. Specifically, I show that: (1) phorbol ester treatment, presumably through the phosphorylation of CD4, increases the association of CD4 with coated pits 3 fold; (2) the rate of CD4 endocytosis is increased 3 fold, and the intracellular pool of CD4 is doubled at equilibrium in the presence of phorbol ester. Fluid-phase internalization is not affected by PMA treatment; (3) in the absence of PMA CD4 is endocytosed into the transferrin receptor-containing early endosomal compartment, from where it can recycle to the plasma membrane. In the presence of phorbol ester however, CD4 is diverted from the early endosome-plasma membrane recycling pathway, to a compartment in the perinuclear region of the cell, that can be costained with antibodies to the cation independent mannose 6- phosphate receptor (CI-MPR); (4) inhibition of kinase and phosphatase activitities, inhibits the internalization and recycling of CD4, respectively, suggesting that the constitutive endocytosis and recycling of CD4 in HeLa-CD4 cells, may involve cycles of phosphorylation and dephosphorylation