Phorbol Ester-induced Disruption of the CD4-Lck Complex Occurs within a Detergent-resistant Microdomain of the Plasma Membrane

Abstract

Recent studies have highlighted the existence of discrete microdomains at the cell surface that are distinct from caveolae. The function of these microdomains remains unknown. However, recent evidence suggests that they may participate in a subset of transmembrane signaling events. In hematopoietic cells, these low density Triton-insoluble (LDTI) microdomains (also called caveolae-related domains) are dramatically enriched in signaling molecules, such as cell surface receptors (CD4 and CD55), Src family tyrosine kinases (Lyn, Lck, Hck, and Fyn), heterotrimeric G proteins, and gangliosides (GM 1 and GM 3 ). Human T lymphocytes have become a well established model system for studying the process of phorbol ester-induced down-regulation of CD4. Here, we present evidence that phorbol 12-myristate 13-acetate (PMA)-induced down-regulation of the cell surface pool of CD4 occurs within the LDTI microdomains of T cells. Localization of CD4 in LDTI microdomains was confirmed by immunoelectron microscopy. PMA-induced disruption of the CD4-Lck complex was rapid (within 5 min), and this disruption occurred within LDTI microdomains. Because PMA is an activator of protein kinase C (PKC), we next evaluated the possible roles of different PKC isoforms in this process. Our results indicate that PMA induced the rapid translocation of cytosolic PKCs to LDTI microdomains. We identified PKC␣ as the major isoform involved in this translocation event. Taken together, our results support the hypothesis that LDTI microdomains represent a functionally important plasma membrane compartment in T cells.

Association of the Death-inducing Signaling Complex with Microdomains after Triggering through CD95/Fas

“…Briefly, 2 ϫ 10 8 cells were suspended in 1 ml of lysis buffer containing 1% Triton-X-100, 10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 1 mM NaVO 4 , and 75 units of aprotinin and allowed to stand for 20 min. The cell suspension was mechanically disrupted by Dounce homogenization (10 strokes).…”

“…Recently, we studied the glycosphingolipid composition of these specialized portions of plasma membrane in human peripheral blood lymphocytes (7), demonstrating by high performance thin layer chromatography, gas chromatographic and mass spectrometric analysis that GM3 represents one of the main markers of microdomains in these cells. Minor components are sialosyl paragloboside (NeuAc-nLac 4 Cer), migrating between GM3 and GM1, sialosyl lactohexaosyl ceramide, and disialoganglioside GD3 (2,10,11). Gangliosides may be involved in modulating signal transduction, mainly by interaction with specific signal transducer molecules detected in these domains (8), such as p56 lck (12) or Zap-70 (7).…”

“…represents the main ganglioside constituent of cell plasma membrane (2), where it is concentrated in glycosphingolipidenriched microdomains (GEM) (3,4). The presence of tyrosine kinase receptors, mono-(Ras, Rap) and heterotrimeric G proteins, Src-like tyrosine kinases (Lck, Lyn, Fyn), PKC isozymes, glycosylphosphatidylinositol-anchored proteins (5,6) and, after T cell activation, the Syk family kinase Zap-70 (7) prompts these portions of the plasma membrane to be considered as "glycosignaling domains" (8,9).…”

Inhibition of the Calcium Release-activated Calcium (CRAC) Current in Jurkat T Cells by the HIV-1 Envelope Protein gp160

“…However, gp160 also induced a 3-fold increase in PKC␤I amount, which resulted in the enrichment of the Triton-insoluble (150%) fraction. Very recently, it has been proposed that the Triton-insoluble fraction contains lipid components of the rafts membrane microdomains (47) and, therefore, might represent membrane-bound protein. Accordingly, Nixon and McPhail (48) have recently shown that upon stimulation the levels of PKC␣ and -␤I increased in the Triton-insoluble fraction.…”

Markers for Detergent-resistant Lipid Rafts Occupy Distinct and Dynamic Domains in Native Membranes

“…The interpretation of gradient centrifugation experiments remains controversial. There is evidence that detergents may force associations between components that are not colocalized in intact cells (Mayor and Maxfield, 1995), and fractionation results are known to be dramatically altered by varying the concentration of Triton X-100 (Field et al, 1999;Parolini et al, 1999), by use of alternative detergents (Montixi et al, 1998;Surviladze et al, 1998) or by omission of detergent altogether (Ilangumaran et al., 1999;Harder and Kuhn, 2000;Surviladze et al, 2001). Methods to observe membrane segregation in situ have also generated controversy.…”