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 Kd values of 0.016 and 9.1 nM, respectively. In the presence of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) 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 GTP gamma S. 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 Mr of 70,000 or over 500,000 in the presence or absence of Mg2+ (5-20 mM), respectively, in solubilizing buffers. The Kd 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 GTP gamma S. The preparation solubilized in the absence of Mg2+ showed both high- and low-affinity binding sites for LTD4, and the addition of GTP gamma S 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.
Watanabe
T
T
First Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Japan.
Shimizu
T
T
Miki
I
I
Sakanaka
C
C
Honda
Z
Z
Seyama
Y
Y
Teramoto
T
T
Matsushima
T
T
Ui
M
M
Kurokawa
K
K
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002850:Chromatography, Gel; D019204:GTP-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006168:Guinea Pigs; D007700:Kinetics; D008168:Lung / Q000378:metabolism*; D015636:Magnesium Chloride / Q000494:pharmacology; D037342:Pertussis Toxin; D011971:Receptors, Immunologic / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D018077:Receptors, Leukotriene; D013189:SRS-A / Q000378:metabolism*; D012995:Solubility; D010566:Virulence Factors, Bordetella / Q000494:pharmacology*
D011971:Receptors, Immunologic; D018077:Receptors, Leukotriene; D013189:SRS-A; D010566:Virulence Factors, Bordetella; D015636:Magnesium Chloride; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D037342:Pertussis Toxin; D019204:GTP-Binding Proteins