Characterization of the guinea pig lung membrane leukotriene D₄ receptor solubilized in an active form: Association and dissociation with an islet-activating protein-sensitive guanine nucleotide-binding protein

Membrane fractions from the guinea pig lung had high- and low-affinity binding sites for LTD₄ with K_d values of 0.016 and 9.1 nM, respectively. In the presence of guanosine 5′-O-(3-thiotriphosphate) (GTPγS) or by prior treatment of the membrane with islet-activating protein (IAP), the high-affinity site shifted to a low-affinity state. Consistently, a 41-kDa protein was ADP-ribosylated by treatment of the lung membranes with IAP, and this event was inhibited by the addition of GTPγS. We solubilized the LTD₄ receptor from the lung membranes in an active form with 5 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and 10% glycerol. On a gel filtration column, the binding activity was eluted at the volume corresponding to a M_r of 70,000 or over 500,000 in the presence or absence of Mg²⁺ (5-20 mM), respectively, in solubilizing buffers. The K_d value of [³H]LTD₄ binding to the 70-kDa protein was similar to the low-affinity binding constant of the membrane and was insensitive to GTPγS. The preparation solubilized in the absence of Mg²⁺ showed both high- and low-affinity binding sites for LTD₄, and the addition of GTPγS shifted the high-affinity site to a low-affinity one. Thus, 1) the LTD₄ receptor is coupled to an IAP-sensitive GTP-binding protein, 2) this GTP-binding protein is dissociable from the receptor by solubilizing the lung membrane with CHAPS and Mg²⁺, and 3) the receptor associated to or dissociated from a GTP-binding protein exhibited a high- or low-affinity state, respectively. These data provide an insight into the molecular mechanism of regulation of the LTD₄ receptor signaling process by association and dissociation with an IAP-sensitive GTP-binding protein.