Characterization of partially purified prostaglandin E2 receptor from the canine renal medulla: evidence for physical association of the receptor protein with the inhibitory guanine nucleotide-binding protein.
Prostaglandin (PG) E2 binding protein, a putative PGE2 receptor, was purified 26-fold with 0.4% recovery from canine renal outer medullary membranes solubilized with 12% digitonin with the sequential use of a Superose 12, Wheat Germ Agglutinin (WGA) Affigel 10, DEAE-5PW and Ampholine column chromatographies. The final preparation retained the binding activity specific for PGE2, but lost most of the sensitivity to guanosine-5'-(gamma-thio)triphosphate (GTP gamma S). An antibody against alpha subunit of the inhibitory guanine nucleotide-binding protein (alpha Gi)1 and alpha Gi2 or that against common sequences of alpha subunit of guanine nucleotide-binding proteins (alpha G(common)) reacted at 41 kDa protein in the sample of each step of purification, but failed to do so in the final preparation. An antibody against alpha Gi3 or alpha Go had no effect. In fact, peaks of the binding activity and immunoreactivity for alpha Gi1,2 were chromatographically separated by isoelectric focusing. Moreover, antibodies against alpha G(common) or alpha Gi1,2, but not that against alpha Gi3 and alpha Go, precipitated PGE2 binding activity in the active fractions of WGA-Affigel 10 column chromatography. These results suggest that the PGE2 receptor is an acidic glycoprotein and that Gi1 or Gi2 is physically associated with the PGE2 receptor and dissociates from the receptor protein during purification procedures.
Watanabe
T
T
First Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Japan.
Shimizu
T
T
Nakao
A
A
Taniguchi
S
S
Arata
Y
Y
Teramoto
T
T
Seyama
Y
Y
Ui
M
M
Kurokawa
K
K
D000818:Animals; D015153:Blotting, Western; D002853:Chromatography, Liquid; D015232:Dinoprostone / Q000378:metabolism*; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D019204:GTP-Binding Proteins / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D007525:Isoelectric Focusing; D007679:Kidney Medulla / Q000378:metabolism*; D011233:Precipitin Tests; D011982:Receptors, Prostaglandin / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D018078:Receptors, Prostaglandin E
D011982:Receptors, Prostaglandin; D018078:Receptors, Prostaglandin E; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D019204:GTP-Binding Proteins; D015232:Dinoprostone