The activation of many cell types involves a multistep pathway initiated by the binding of ligand (L) to cell surface receptors (R) and followed by a transmembrane interaction of LR with guanine nucleotide binding proteins (G). The neutrophil, whose G is a novel protein which activates phospholipase C, is an ideal model system in which to study LRG dynamics. Neutrophils, the most numerous of the human white blood cells, play a pivotal role in host defense and contribute to tissue injury which accompanies inflammation. Fluorescent ligands (both agonists and antagonists) for several receptors have already been prepared. The ligands together with homogeneous, real-time, fluorescent methods make possible for the first time a detailed analysis of LRG dynamics and its role in cell activation. The formyl peptide receptor of the neutrophil has already been identified in three states: 1) a ternary LRG complex which is slowly dissociating for L and sensitive to the presence of guanine nucleotide; 2) a rapidly dissociating LR complex; and 3) a distinct, slowly dissociating state insensitive to guanine nucleotide which is hypothesized to arise subsequent to cell activation. During cell activation there appears to be interconversion among these forms, with the first and perhaps the second active in transduction. These states have unique biochemical characteristics and can be studied individually. In order to understand 1) the roles of these states, 2) the dynamics of their interconversion, and 3) their interactions with G proteins during cell activation, we will first define the conditions of formation, stabilization and interconversion among the states in cell and membrane preparations, and where possible, with pure proteins. Then we will define the rate processes and thermodynamics associated with the states and their interconversions. We will use pulse stimulation and rapid kinetic techniques to assess the appearance and disapperance of the states during cell activation. We will use mathematical modelling to gain mechanistic insight into the transduction pathway. By comparing results for different agonists we expect to gain fundamental insight into the basis of agonist efficacy. By comparing the agonists and antagonists we expect to define fundamental differences in the ability of agonists or antagonists to interact with or promote interconversion among receptor states. By contrasting results obtained with formyl peptides and fluorescent C5a we expect to gain insight into the uniqueness or generality of LRG dynamics at receptors for distinct ligands.
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