Solubilization of high affinity corticotropin-releasing factor receptors from rat brain: characterization of an active digitonin-solubilized receptor complex.
The binding characteristics of CRF receptors in rat frontal cerebral cortex membranes solubilized in 1% digitonin were determined. The binding of [125I]Tyro-ovine CRF ([125I]oCRF) to solubilized membrane proteins was dependent on incubation time, temperature, and protein concentration, was saturable and of high affinity, and was absent in boiled tissue. The solubilized receptors retained their high affinity for [125I] oCRF in the solubilized state, exhibiting a dissociation constant (KD) of approximately 200 pM, as determined by direct binding saturation isotherms. Solubilized CRF receptors maintained the rank order of potencies for various related and unrelated CRF peptides characteristic of the membrane CRF receptor: rat/human CRF congruent to ovine CRF congruent to Nle21,38-rat CRF greater than alpha-helical oCRF-(9-41) greater than oCRF-(7-41) much greater than vasoactive intestinal peptide, arginine vasopressin, or the substance-P antagonist. Furthermore, the absolute potencies (Ki values) for the various CRF-related peptides in solubilized receptors were almost identical to those observed in the membrane preparations, indicating that the CRF receptor retained its high affinity binding capacity in the digitonin-solubilized state. Chemical affinity cross-linking of digitonin-solubilized rat cortical membrane proteins revealed a specifically labeled protein with an apparent mol wt of 58,000 which was similar to the labeled protein in native membrane homogenates. Although solubilized CRF receptors retained their high affinity for agonists, their sensitivity for guanine nucleotide was lost. Size exclusion chromatography substantiated these results, demonstrating that in the presence or absence of guanine nucleotides, [125I]oCRF labeled the same size receptor complex. These data suggest that either the guanine nucleotide-binding protein (Ns) is tightly associated with the CRF receptor after solubilization and is insensitive to guanine nucleotides, or that high affinity binding for soluble CRF receptors is not dependent on the coupling of a guanine nucleotide-binding protein. The solubilization of CRF receptors from membranes in digitonin should allow for the more complete molecular and functional characterization of CRF-mediated events and purification of the receptor.
Grigoriadis
D E
DE
Laboratory of Neurobiology, Neuroscience Branch, National Institute on Drug Abuse, Baltimore, Maryland 21224.
Zaczek
R
R
Pearsall
D M
DM
De Souza
E B
EB
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003346:Corticotropin-Releasing Hormone / Q000378:metabolism / Q000031:analogs & derivatives; D003902:Detergents; D004072:Digitonin; D005625:Frontal Lobe / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate / Q000494:pharmacology; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D007457:Iodine Radioisotopes; D008297:Male; D008970:Molecular Weight; D011092:Polyethylene Glycols; D051381:Rats; D011919:Rats, Inbred Strains; D018019:Receptors, Corticotropin-Releasing Hormone; D017981:Receptors, Neurotransmitter / Q000378:metabolism*; D012995:Solubility; D013873:Thionucleotides / Q000494:pharmacology
D003902:Detergents; D007457:Iodine Radioisotopes; D018019:Receptors, Corticotropin-Releasing Hormone; D017981:Receptors, Neurotransmitter; D013873:Thionucleotides; C061959:corticotropin-releasing hormone, iodo-Tyr(0)-; D006165:Guanylyl Imidodiphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D011092:Polyethylene Glycols; D006160:Guanosine Triphosphate; D003346:Corticotropin-Releasing Hormone; D004072:Digitonin