Molecular Bar Coding and Cell Specific Signaling with Multivalent Tethered Ligands

Targeting cells with ligands to a single surface receptor does not provide cell specific labeling due to promiscuity of receptor expression. However, targeting a combination of surface markers (conceptually bar coding) may greatly enhance specificity. We developed a multivalent ligand (MVL) by linking melanocyte stimulating hormone (MSH) to cholecystokinin (CCK), then evaluated its binding, specificity and activity. In cells with both receptors, the Kd of the MLV was ~5 nM compared to > 250 nM when one receptor was blocked with cold MSH (i.e., monvalent CCK binding). Moreover, at concentrations <5 nM, binding to cells with only one receptor was negligible, demonstrating high specificity. MVL activation of the second messengers cAMP (MSH) and Ca 2+ (CCK) was tested. Even though the MVL has enhanced affinity, both the cAMP and Ca 2+ responses were attenuated compared to the monovalent ligands. Importantly, in cells expressing only CCKR, the increase in Ca 2+ elicited by the MVL also was decreased compared to CCK suggesting that cross talk between CCK and MC4 receptors is not responsible for the lower efficacy of the CCK moiety. Though less effective, the ability of the MVL to activate signaling is further evidence that the MLV binds to and activates both receptors. Moreover, the high selectivity and binding affinity coupled with altered signaling may be a useful pharmacological property of these new multivalent ligands. Supported by NIH, CA097360, CA097360.