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Prostaglandin E2 (PGE2) was found to bind specifically, reversibly, and in a protein-dependent manner to a single class of high affinity (KD approximately equal to 20 nM) binding sites in membranes prepared from canine renal outer medulla. PGE2 binding activity was solubilized from these membranes in a stable form (t1/2 greater than 14 days) in the absence of ligand in 75% yields using digitonin. The characteristics of PGE2 binding to membranes and solubilized protein were similar with respect to pH dependence, KD for PGE2, and order of potency of prostaglandins (PGE2 approximately PGE1 greater than PGF2 alpha greater than PGD2) in inhibiting the binding of [3H]PGE2. Importantly, the extents of binding of PGE2 to membranes and to a solubilized preparation partially purified by chromatography on wheat germ agglutinin-Affi-Gel 10 were both increased about 2-fold by GDP and GTP and its analogs. Treatment of the digitonin-solubilized PGE2 binding activity with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS) rendered the binding activity insensitive to stimulation by GTP and decreased the apparent molecular weight of the peak of PGE2 binding activity from about 175,000 to about 65,000. These results suggest that the PGE2 binding activity resides in a protein which is tightly associated with, but distinct from, a guanine nucleotide regulatory (N) protein. PGE2 (greater than or equal to 10 nM) was found to stimulate GTPase activity of renal outer medullary membranes, and this stimulation was eliminated by pretreatment of membranes with pertussis toxin and NAD, but not cholera toxin and NAD. Treatment of both particulate and solubilized preparations of PGE2 binding activity with pertussis toxin plus NAD also eliminated the ability of GTP to stimulate PGE2 binding. This evidence indicates that it is the inhibitory guanine nucleotide regulatory protein, Ni, with which the PGE2 binding activity is associated. Thus, this PGE2 binding activity is an inhibitory PGE2 receptor, quite possibly one that mediates inhibition of vasopressin-induced cAMP formation in the medullary thick ascending limb and/or collecting tubule of the kidney.
T Watanabe, and
K Umegaki, and
W L Smith
September 1987,
The Journal of biological chemistry,
T Watanabe, and
K Umegaki, and
W L Smith
February 1991,
Journal of biochemistry,
T Watanabe, and
K Umegaki, and
W L Smith
August 1991,
Biochimica et biophysica acta,
T Watanabe, and
K Umegaki, and
W L Smith
May 1990,
The Journal of biological chemistry,
T Watanabe, and
K Umegaki, and
W L Smith
March 1989,
The Journal of pharmacology and experimental therapeutics,
T Watanabe, and
K Umegaki, and
W L Smith
July 1991,
The Journal of biological chemistry,
T Watanabe, and
K Umegaki, and
W L Smith
May 1988,
The Journal of biological chemistry,
T Watanabe, and
K Umegaki, and
W L Smith
July 1986,
Biochemical and biophysical research communications,
T Watanabe, and
K Umegaki, and
W L Smith
August 1993,
The Journal of pharmacology and experimental therapeutics,
T Watanabe, and
K Umegaki, and
W L Smith
August 1989,
Nihon Naibunpi Gakkai zasshi,
