RESULTADOS RESPECTO AL MINISTERIO DE COMERCIO EXTERIOR Y

l.T.a Activation O f Protein Kinase C.

Protein kinase C (PKC) is activated by the recep to r-m ed iated hydrolysis of inositol phospholipids by phospholipase C (PLC), it is responsible for the phosphorylation of a num ber o f intracellular s u b s tr a te s and fo r r e la y in g in f o r m a tio n , in the fo rm o f

e x tr a c e llu la r sig n als, across the p lasm a m em b ran e to reg u la te m an y Ca^+-dependent events (reviewed by N ishizuka 1986).

T he prim ary products of PLC hydrolysis of phosphotidylinositol 4 ,5 - b is p h o s p h a te ( P I P2), are inositol 1,4,5-triphosphate (IP3), and 1,2-diacylglycerol (DAG) (Im boden and Stobo 1985; Pantaleo et al 1987). T h ese p ro d u cts act as seco n d ary m essen g ers. IP3 is resp o n sib le for the release o f Ca^+ fro m in tra c e llu la r stores, w hilst D A G is the physiological activator of PKC.

Phorbol esters, such as phorbol myristic acid (PMA), have a very sim ilar structure to D A G and are capable o f activating PKC both in v itr o and in vivo (Castagna et al 1982). Phorbol esters have been show n to affect the normal cellular distribution and trafficking of a variety of receptors including TfR, EGF-R, ASGP-R and LDL-R, w ithin certain cell types (M cG raw et al 1988; Magun et al 1980; Fallon and Schwartz 1987; M aziere et al 1986), and have also been shown to activate the N a+/H + exchange protein (B esterm an et al 1985). R ecen tly p h o rb o l esters have been show n to stim ulate transcytosis in M D C K cells (C ardone et al 1994), and constitutive secretion in rat basophilic leukaemia cells (De Matteis et al 1993).

In addition to these effects, phorbol esters have m ore general e f f e c ts on c e ll a r c h ite c tu r e and m e m b ra n e p r o c e s s e s . In p a rticu lar, p h orbol esters induce dram atic changes in cell shape and m em brane in fibroblasts and m acrophages (M iyata et al 1988; P h a ire -W a s h in g to n et al 1980a). PMA affects the organization of m i c r o t u b u l e s and m i c r o f i l a m e n t s in m a c r o p h a g e s ( P h a ir e - W a sh in g to n et al 1980b), which may account for the changes in cell shape, and stim ulation of fluid-phase uptake and delivery to lysosom es (Sw anson et al 1 9 S 5 ).

Therefore, with these potential effects in mind, interpretation of results when em ploying phorbol esters can be very difficult, and require the use of stringent control experiments. D espite these e ffects, p h o rb o l esters have proved to be an e x trem ely useful reagent in the study of T cell activation, and are known to cause the modulation of CD4.

l.T.b Dow n-Regula ti on O f CD4 A n d Its Intera ction With The E ndocytic Pathway.

HIV. Until recently little information was available concerning its endocytic properties and intracellular trafficking. The cell surface expression of CD4 is correlated with activation of T cells, such that c ell surface CD4 is m odulated follow ing exposure o f T cells to specific antigen (Acres et al 1986; W eyand et al 1987; Rivas et al 1988), or to cross-linking antibodies the TCR/CD3 complex (Rivas et a l 1988), or CD2 (Blue et a l 1989). In addition, the HIV early p ro te in , N e f (A iken et al 1994), and c ro s s-lin k in g a n tib o d ie s a g ain st CD4 (L edbetter et al 1988; Cole et al 1989; Thuillier et al 1990), cause down-regulation of CD4. The down-regulation of CD4 observed in response to antigenic stimulation o f T cells, can be m im ick ed by treatm ent of cells with phorbol esters (Acres et al 1986; W eyand et al 1987), which activate PKC (Nishizuka 1986), and cause the transient phosphorylation o f the CD4 cytoplasm ic dom ain (Acres et al 1986; Blue et al 1987; Hoxie et al 1988). The ex ac t m echanism by which cell surface CD4 is dow n-regulated is n o t fu lly un d ersto o d , but it is th ought th at it m ay o ccu r by e n d o c y t o s i s (H o x ie et al 1986; 1988; Petersen et al 1992), fo llo w in g the d isso c ia tio n o f p56^^^ ( P e l c h e n - M a t t h e w s et al 1993), and there is some indication in the literature that CD4 is degraded follow ing phorbol ester treatm ent (Baenziger et al 1991; Shin et al 1991; Petersen et al 1992; Ruegg et al 1992). Despite these reports, the interaction of CD4 with the endocytic pathw ay and its ex act intracellular fate during dow n-regulation have not b e e n d e te rm in e d .

P re v io u s resu lts on the end o cy tic p ro p erties o f h um an CD4 expressed in non-lymphoid cells (HeLa-CD4 and NIH3T3-CD4) and m o n o c y t i c c e l ls ( H L - 6 0 ) h a v e d e m o n s t r a t e d t h a t it is co n stitu tiv e ly endocytosed into the early endosom e and recycled to the cell surface (Pelchen-M atthew s et al 1989; 1991; Marsh e t a l 1990). Internalization occurs through clathrin-coated pits and vesicles, and at steady state about 40% of the CD4 is found inside the cells. T he rates of CD4 internalization in H eLa-C D 4 and N IH 3T 3-C D 4 cells (2-3% per min and 4% per min, respectively), are sig n ific an tly faster than b ulk-flow uptake of m u ta n t CD4 m o lecu les lacking a cytoplasm ic dom ain (Pelchen-M atthew s et al

1991). In contrast, CD4 expressed in lym phocytic cells is not e n d o c y to se d (P e lch e n -M atth ew s et al 1991), due to its association w ith p56^<^^ which prevents its entry into cla th rin -co a ted pits