LIGAND RECEPTOR MODELS SYSTEMS

University Of Calif-Los Alamos Nat Lab, Los Alamos NM

Investigators

Linked publications & trials

Abstract

The aim of the project is to characterize the interactions of the formyl peptide ligands with formyl peptide receptors and to use these peptides as models for 1) developing the rapid mix flow cytometer and 2) studying signal transduction. A family of formyl peptides with fluorescein isothiocyanate conjugated at positions 2 through 7 has been generated and their binding to receptors has been characterized by flow cytometry (Vilven et al.1999). Prossnitz and Ye have produced mutant receptors whose kinetics are being characterized in the rapid mix flow cytometer (Prossnitz et al. 1999). The molecular assemblies, receptor processing, and desensitization of these receptors are being evaluated. The data suggest that receptors exist in two new forms: the native form which is different from that detected in cell membrane preparations (Gilbert et al. 1999), and a continuously active receptor form which is not yet phosphorylated but is nonetheless beginning to be regulated in the signal transduction pathway. A kinetic analysis of the mutant receptors which are not phosphorylated reveals the formation of a novel receptor state attributed to a continuously active form (Gilbert et. al, in preparation). We used Freer's peptides to probe the role of ligand affinity in the activity and kinetics of the novel receptor forms. We have begun to examine both receptors and signaling molecules displayed on beads for flow cytometry (Nolan and Sklar, 1998; Sklar et al. in preparation). We have shown conditions under which his-tagged receptors may be bound to Ni2+ beads and bind ligand. We have also shown that solublized receptors may be reconstituted with heterotrimeeric G proteins for fluorescence, and potentially for flow cytometric analysis. The first Research Highlight in this report describes an aspect of this effort.