Cluster of differentiation antigen 4 (CD4), the T lymphocyte antigen receptor component and human immunodeficiency virus coreceptor, is down-modulated when cells are activated by antigen or phorbol esters. During down-modulation CD4 dissociates from p56 lck , undergoes endocytosis through clathrin-coated pits, and is then sorted in early endosomes to late endocytic organelles where it is degraded. Previous studies have suggested that phosphorylation and a dileucine sequence are required for down-modulation. Using transfected HeLa cells, in which CD4 endocytosis can be studied in the absence of p56 lck , we show that the dileucine sequence in the cytoplasmic domain is essential for clathrin-mediated CD4 endocytosis. However, this sequence is only functional as an endocytosis signal when neighboring serine residues are phosphorylated. Phosphoserine is required for rapid endocytosis because CD4 molecules in which the cytoplasmic domain serine residues are substituted with glutamic acid residues are not internalized efficiently. Using surface plasmon resonance, we show that CD4 peptides containing the dileucine sequence bind weakly to clathrin adaptor protein complexes 2 and 1. The affinity of this interaction is increased 350- to 700-fold when the peptides also contain phosphoserine residues.

The two clathrin-associated adaptor complexes AP1 and AP2 are known to participate in the formation of clathrin-coated vesicles at the trans-Golgi network and at the plasma membrane. During this process adaptors are involved in the sequestration of vesicle cargo by binding to the sorting signals within the cytoplasmic domains of the cargo proteins and in the recruitment of the clathrin coat. After budding of the clathrin-coated vesicles, the clathrin and adaptors dissociate from the vesicles. Here we show that in vitro binding of AP2 to sorting signals, which is one of the initial steps in receptor-mediated endocytosis, is modulated by adaptor phosphorylation. AP2 was phosphorylated by incubating purified AP2 in the presence of ATP and dephosphorylated by incubation with alkaline phosphatase. Affinity for tyrosine-, leucine-based and noncanonical sorting motifs was 15-33 times higher for phosphorylated than for dephosphorylated AP2. Also the binding of AP2 to membranes was regulated by adaptor phosphorylation/dephosphorylation and was about 8-fold higher for phosphorylated than for dephosphorylated AP2. Moreover, AP2 isolated from cytosol is higher phosphorylated than membrane-extracted and exhibits a 5-fold higher binding affinity than AP2 extracted from membranes. Taken together these data point to a cycle of phosphorylation/dephosphorylation as a mechanism for regulating the reversible association of AP2 with membranes and sorting signals during the process of receptor-mediated endocytosis.Trafficking of membrane proteins within the secretory and endocytic route of eukaryotic cells is characterized by the sorting of the membrane proteins at sites where transport pathways diverge and the packaging of the membrane proteins into vesicles occurs. The adaptors AP1 and AP2 appear to play a key role in the sorting and packaging of membrane proteins in clathrin-coated vesicles (CCVs). 1 AP1-containing CCVs are formed at the TGN and facilitate the transport of cargo from the TGN to endosomes, whereas AP2-containing CCVs function in receptor-mediated endocytosis at the plasma membrane. The adaptors are heterotetrameric complexes composed of two 100-kDa subunits (designated ␥ and ␤1 in AP1 and ␣ and ␤2 in AP2; for review see Ref. 1). The ␣-, ␤1-, and ␤2-subunits have been implicated in clathrin binding (2-4). Recognition of the membrane proteins is mediated by the medium subunits 1 and 2 (5), but also the ␤-subunits have been implicated in binding to membrane targets (6, 7). Tyrosine-and leucinebased sorting motifs in cytoplasmic tails of membrane proteins can serve as adaptor binding sites, and the tyrosine and leucine residues have been shown to be critical for the sorting of the membrane proteins, as well as for the adaptor binding (for review see Ref. 8). For noncanonical sequences mediating either sorting or adaptor binding, this correlation, however, remains to be established. After formation of the CCVs, the clathrin coat is removed by the uncoating ATPase (hsc70) in a reaction depending on ATP hydrolysis (9 -11)...

The Pro12Ala polymorphism of the PPARgamma gene is associated with increased insulin sensitivity and lower hirsutism scores in PCOS women.

This review presents non-heme iron catalyzed epoxidations and aziridinations of terminal alkenes with respect to atom economy and sustainability.