Affinity purification of 61- and 65-kDa rat brain corticotropin-releasing factor receptors and receptor-associated G proteins.
Corticotropin-releasing factor (CRF) has been shown to have a central role in physiological adaptation to stress. It is recognized for stimulating the release of adrenocorticotropin from the anterior pituitary gland, and has more recently been implicated as a regulator of autonomic and immunological responses to stress. Much confusion has surrounded the characterization of CRF receptors, with proteins of varying molecular weights having been identified but never purified and characterized. Recently, two CRF receptors have been cloned from brain and pituitary gland, but evidence from in-situ hybridization studies suggests that further CRF receptor types exist. We therefore developed two techniques which enable the isolation of CRF receptors from whole rat brain. The use of a solid-phase CRF analogue affinity column and elution using a competing ligand resulted in the purification of a single protein of 61 kDa. A second technique was devised which allowed the co-isolation of associated signalling proteins and the identification of CRF bound species following purification. CRF was covalently cross-linked to receptors and the complex purified using antibodies specific for the ligand. This enabled the purification of a CRF receptor of approximately 65 kDa and associated alpha and beta gamma G protein subunits. This study demonstrates the successful isolation of CRF receptors which are of different molecular weights to those previously observed from affinity cross-linking studies or predicted from cloned genes. In addition, we confirm the involvement of G proteins in CRF stimulated cell signalling by demonstrating their association with purified CRF receptor.
Gibbins
J M
JM
School of Animal and Microbial Sciences, University of Reading, Whiteknights, U.K. jonathan.gibbins@pharmacology.ox.ac.uk
Woods
R J
RJ
Lowry
P J
PJ
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001923:Brain Chemistry; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D051381:Rats; D018019:Receptors, Corticotropin-Releasing Hormone / Q000302:isolation & purification* / Q000378:metabolism; D012756:Sheep; D015398:Signal Transduction
D000596:Amino Acids; D018019:Receptors, Corticotropin-Releasing Hormone; D019204:GTP-Binding Proteins