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 stimu lating the release of adrenocorticotropin from the anterior pituitary gland, and has more recently been implicated as a regulator of autonom ic and immunological responses to stress. Much confusion has surrounde d the characterization of CRF receptors, with proteins of varying mole cular weights having been identified but never purified and characteri zed. Recently, two CRF receptors have been cloned from brain and pitui tary gland, but evidence from in-situ hybridization studies suggests t hat further CRF receptor types exist. We therefore developed two techn iques which enable the isolation of CRF receptors from whole rat brain . The use of a solid-phase CRF analogue affinity column and elution us ing a competing ligand resulted in the purification of a single protei n of 61 kDa. A second technique was devised which allowed the co-isola tion of associated signalling proteins and the identification of CRF b ound species following purification. CRF was covalently cross-linked t o receptors and the complex purified using antibodies specific for the ligand. This enabled the purification of a CRF receptor of approximat ely 65 kDa and associated alpha and beta gamma G protein subunits. Thi s study demonstrates the successful isolation of CRF receptors which a re of different molecular weights to those previously observed from af finity cross-linking studies or predicted from cloned genes. In additi on, we confirm the involvement of G proteins in CRF stimulated cell si gnalling by demonstrating their association with purified CRF receptor . Copyright (C) 1996 Elsevier Science Ltd