Platelet-activating factor (PAF) is a unique lipid mediator of acute allergic an inflammatory reactions that elicits a powerful and exceptionally potent stimulatory effect on hepatic glycogenolysis by an indirect mechanism involving constriction of the hepatic vasculature. Our findings that PAF regulates hepatic glycogenolysis and blood flow in vivo and during antigen-induced anaphylaxis suggests a crucial role for PAF in supplying extrahepatic tissues with energy substrates and regulating the hepatic microcirculation during acute pathophysiologic situations. The proposed research will carry our studies of the role of PAF in liver pathology to the molecular level and provide the detailed information needed to advance this area. Using a PCR-based strategy, we successfully cloned cDNAs encoding the rat liver PAF R, lacking only the extreme 5' coding region of the PAF R cDNA. Sequence analysis of the hepatic PAF R demonstrates that it is a member of the G protein-coupled receptor family and differs from the recently cloned leukocyte PAF R in intracellular domains implicated in the signaling actions of several other G protein- coupled receptors. We also amplified a truncated form of the PAF R cDNA, encoding a protein lacking a C terminal cytoplasmic domain, providing the first evidence for the existence of an alternate short form of the PAF R. In situ hybridization revealed prominent expression of the PAF R mRNA in hepatic sinusoidal cells which our studies have implicated as the primary site of PAF action in liver. The availability of full length PAF R cDNAs will enable us to pursue detailed studies to address questions relating to PAF R heterogeneity, mechanisms for regulation of PAF R gene expression and elucidating the structure and function of receptor domains of the PAF R to provide new insights into the molecular basis of PAF action in liver. In the proposed studies, we will 1) amplify the remaining 5' end of the short and long PAF R cDNAs or alternatively, screen a rat liver cDNA library, to obtain the full length cDNAs encoding these two forms of the PAF R. We will 2) utilize transient expression of the these PAF R cDNAs to determine the ligand binding, signaling and desensitization properties of these two PAF Rs, including identification of the G proteins activated by the receptors. A major focus of this project will be to 3) evaluate the structure and function of intracellular domains of the receptor in signaling add desensitization in studies utilizing coexpression, truncation and site-directed mutations of these receptor domains. Finally, we will 4) identify the hepatic cells expressing the PAF R mRNA, determine whether there is differential expression of the short and long forms of the PAF R mRNA and assess whether hepatic responses to PAF are regulated at the transcriptional level by PAP and endotoxin. These studies will provide new insights into the molecular basis of PAF action in liver. Because no studies have evaluated the molecular basis of PAF R function at the level of the receptor, it is likely that concepts relevant to understanding the molecular basis of PAF action in other tissues will emerge from this work.
