Abstract
Platelet activating factor (PAF) is a potent phospholipid mediator which elicits a diverse array of biological actions by interacting with G protein-coupled PAF receptors (PAFR). Binding of PAF to PAFRs leads to activation of G protein(s) that stimulate phosphoinositide phospholipase C and subsequent intracellular signaling responses. To identify the potential role of intracellular domains of the rat PAFR (rPAFR) in signaling, we examined effects of transfecting minigenes encompassing rPAFR intracellular domains 1 (1i), 2 (2i), and 3 (3i) on inositol phosphate (IP) production mediated by the co-transfected rPAFR cDNA. Although transfection of the rPAFR1i and rPAFR2i minigenes had no effects on PAF-stimulated signaling, transfection of the rPAFR3i minigene inhibited PAF-stimulated IP production by approximately 50% compared to controls. The rPAFR3i domain did not inhibit IP production mediated by the multifunctional rat pituitary adenylate cyclase-activating polypeptide receptor (rPACAPR), demonstrating the specificity of the competition by the rPAFR3i domain. In further experiments, the rPAFR3i domain was engineered onto the homologous domain of a monofunctional transmembrane variant of the rPACAPR (rPACAPR2) that activates only adenylyl cyclase. The rPACAPR2/rPAFR3i chimera responded to PACAP with increases in IP production which were attenuated nearly completely in cells cotransfected with the rPAFR3i domain. In contrast, PACAP had no effects on IP production in a receptor chimera expressing a mutated form of the rPAFR3i domain (rPACAPR2/rPAFR3imut). These results demonstrate the ability of the rPAFR3i domain to confer a phospholipase C-signaling phenotype to a receptor deficient in this activity and show that this activity is specific for the engineered rPAFR3i domain. These results suggest that the third intracellular loop of the rPAFR is a primary determinant in its coupling to phosphoinositide phospholipase C-activating G proteins, providing the first insight into the molecular basis of interaction of PAFRs with signal-transducing G proteins.
Highlights
Platelet activating factor (PAF) produces its biological effects by interacting with specific receptors, identified and characterized in numerous cells and tissues [9, 10]
These results provide the first insight into the structural determinants of the PAF receptors (PAFR) that are involved in its coupling to G proteins
We transfected minigenes encoding the three intracellular loops of the rat PAFR (rPAFR) to antagonize rPAFRmediated inositol phosphate (IP) production to identify potential intracellular regions of the receptor required for coupling to phospholipase C-activating G proteins. This approach, first described by Luttrell et al [25], relies upon disruption of receptor/G protein coupling by competition of intracellular domain peptides with the ligand-receptor complex at the receptor/G protein interface. These studies were complemented by a gain-of-function strategy in which one of the intracellular loops of the rPAFR was engineered onto a receptor deficient in signaling via phospholipase C in an attempt to confer the phospholipase C-signaling phenotype
Summary
PACAP had no effects on IP production in a receptor chimera expressing a mutated form of the rPAFR3i domain (rPACAPR2/rPAFR3imut) These results demonstrate the ability of the rPAFR3i domain to confer a phospholipase C-signaling phenotype to a receptor deficient in this activity and show that this activity is specific for the engineered rPAFR3i domain. We used a combination of minigene expression of intracellular domains of the rPAFR to antagonize PAFR-mediated signaling and a novel gain of function approach using insertional receptor chimerogenesis to demonstrate that the rPAFR3i plays a crucial role in rPAFR signaling These results provide the first insight into the structural determinants of the PAFR that are involved in its coupling to G proteins
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