Receptor clustering by multivalent ligands can activate signaling pathways. In principle, multivalent ligand features can control clustering and the downstream signals that result, but the influence of ligand structure on these processes is incompletely understood. Using a series of synthetic polymers that vary systematically, we studied the influence of multivalent ligand binding epitope density on the clustering of a model receptor, concanavalin A (Con A). We analyze three aspects of receptor clustering: the stoichiometry of the complex, rate of cluster formation, and receptor proximity. Our experiments reveal that the density of binding sites on a multivalent ligand strongly influences each of these parameters. In general, high binding epitope density results in greater numbers of receptors bound per polymer, faster rates of clustering, and reduced inter-receptor distances. Ligands with low binding epitope density, however, are the most efficient on a binding epitope basis. Our results provide insight into the design of ligands for controlling receptor-receptor interactions and can be used to illuminate mechanisms by which natural multivalent displays function.
The affinities of the carbohydrate-binding protein concanavalin A (Con A) for mono- and multivalent
ligands were measured by surface plasmon resonance (SPR) detection. Assessing protein−carbohydrate affinities
is typically difficult due to weak affinities observed and the complications that arise from the importance of
multivalency in these interactions. We describe a convenient method to rapidly evaluate the inhibitory constants
for a panel of different ligands, both monovalent and multivalent, for low-affinity receptors, such as the
carbohydrate-binding protein Con A. A nonnatural, mannose-substituted glycolipid was synthesized, and self-assembled monolayers of varying carbohydrate density were generated. The synthetic surfaces bind Con A.
Competition experiments that employed monovalent ligands in solution yielded K
i values similar to equilibrium
binding constants obtained in titration microcalorimetry experiments. In addition, this assay could be used to
examine various polymeric ligands of defined lengths, generated by ring-opening metathesis polymerization
(ROMP). This study demonstrates the utility of this method for rapidly screening ligands that engage in low
affinity interactions with their target receptors. Our results emphasize that those molecules that can
simultaneously occupy two or more saccharide binding sites within a lectin oligomer are effective inhibitors
of protein−carbohydrate interactions.
During chemotaxis, activation of the small guanosine triphosphatase Rac is spatially regulated to organize the extension of membrane protrusions in the direction of migration. In neutrophils, Rac activation is primarily mediated by DOCK2, an atypical guanine nucleotide exchange factor. Upon stimulation, we found that DOCK2 rapidly translocated to the plasma membrane in a phosphatidylinositol 3,4,5-trisphosphate–dependent manner. However, subsequent accumulation of DOCK2 at the leading edge required phospholipase D–mediated synthesis of phosphatidic acid, which stabilized DOCK2 there by means of interaction with a polybasic amino acid cluster, resulting in increased local actin polymerization. When this interaction was blocked, neutrophils failed to form leading edges properly and exhibited defects in chemotaxis. Thus, intracellular DOCK2 dynamics are sequentially regulated by distinct phospholipids to localize Rac activation during neutrophil chemotaxis.
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