To analyze the interaction of sorting signals with clathrin‐associated adaptor complexes, we developed an in vitro assay based on surface plasmon resonance analysis. This method monitors the binding of purified adaptors to immobilized oligopeptides in real time and determines binding kinetics and affinities. A peptide corresponding to the cytoplasmic domain of wild‐type influenza hemagglutinin, an apical membrane protein that is not endocytosed, did not significantly bind adaptor complexes. However, peptide sequences containing a tyrosine residue that has previously been shown to induce endocytosis and basolateral sorting were specifically recognized by adaptor complexes. The in vitro rates of adaptor association with these peptides correlated with the internalization rates of the corresponding hemagglutinin variants in vivo. Binding was observed both for purified AP‐2 adaptors of the plasma membrane and for AP‐1 adaptors of the Golgi, with similar apparent equilibrium dissociation constants in the range 10(‐7)‐10(‐6) M. Adaptor binding was also demonstrated for a sequence containing a C‐terminal di‐leucine sequence, the second major motif of endocytosis/basolateral sorting signals. These results confirm the concept that interaction of cytoplasmic signals with plasma membrane adaptors determines the endocytosis rate of membrane proteins, and suggest the model that clathrin‐coated vesicles of the trans‐Golgi network are involved in basolateral sorting.

Monocyte chemoattractant protein-1 (MCP-1) is a chemotactic cytokine mainly acting on monocytes and T cells that elicits its biological effects by interacting with the seven-transmembrane helix receptor CCR2B. The vaccinia virus strain Lister and many other poxviruses express soluble proteins (vCCI) that bind MCP-1 and other CC chemokines and inhibit their function. In order to define the interaction site of MCP-1 with vCCI from vaccinia, surface exposed residues of MCP-1 were identified and mutated to alanine. The MCP-1 variants were expressed, purified, and their interaction with vCCI was characterized. The site on MCP-1 for vCCI binding is dominated by arginine 18 with important additional contributions from tyrosine 13 and arginine 24. These residues define a binding site that largely overlaps with the CCR2B receptor interaction site. The viral chemokine-binding protein vCCI thus inhibits the biological function of MCP-1 by directly masking its CCR2B receptor-binding site.Chemokines are small (8 -14 kDa) structurally related proteins that regulate cell trafficking of various leukocyte subtypes through interaction with a set of G protein-coupled receptors. The two major subfamilies are the CXC chemokines which act on neutrophils and non-hemopoietic cells and the CC chemokines which bind to receptors mainly expressed on monocytes, T cells, eosinophils, and basophils. Additional members of the chemokine family are the C chemokine lymphotactin and the CX3C chemokine fraktalkine. Chemokines are important for the development, homeostasis, and function of the immune system and play a pivotal role in host defense (for reviews, see Refs. 1-3).Given the importance of chemokines for defense against pathogens it is no surprise that viruses themselves developed mechanisms to neutralize the function of chemokines to further their own propagation or evade host defense (4). Poxviruses express secreted chemokine neutralizing proteins termed virus-encoded chemokine-binding proteins (vCKBP).1 Two families of vCKBPs have thus far been identified that use distinct mechanisms of chemokine neutralization (5). The vCKBP-1 family binds C, CC, and CXC chemokines with low affinity, possibly by interaction with the proteoglycan-binding site on chemokines, thereby interfering with the proper localization and presentation of chemokines in inflamed tissues (6, 7). The vCKBP-2 proteins, however, bind preferably CC chemokines with high affinity and inhibit the interaction of chemokines with their cellular receptors (8 -10). vCKBP-2 proteins have been identified in strains of vaccinia (11), cowpox (12), rabbitpox (13), myxomavirus (14), and variola virus (15). Prototypic members of this protein family are the M-T1 gene product from myxoma virus and the rabbitpox virus major secreted 35-kDa protein (10). In rabbits infected with a rabbitpox virus which had the gene for the secreted 35-kDa protein deleted, an increased leukocyte influx into virus-infected tissues was observed, confirming the role of this protein in inhibition of chemokine-medi...

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