Selective impairment of corticotropin-releasing factor(1) (CRF1) receptor-mediated function using CRF coupled to saporin

CRF is the main component in the brain neuropeptide effector system respons ible for the behavioral, endocrine, and physiological activation that accom panies stress activation. Reduced CRF system activation plays a role in the etiology of a variety of psychiatric and metabolic disease states. We have developed a novel protein conjugate that joins native rat/human CRF to a r ibosome-inactivating protein, saporin (CRF-SAP), for the purpose of targete d inactivation of CRF receptor-expressing cells. Cytotoxicity measurements revealed that CRF-SAP (1-100 nM) produced concentration-dependent and progr essive cell death over time in CRF1 receptor-transfected L cells, but at si milar concentrations had no effect on CRF2 alpha receptor-transfected cells . The CRF-SAP-induced toxicity in CRF1-transfected cells was prevented by c oincubation with the competitive CRF1/CRF2 receptor peptide antagonist, [D- Phe(12)] CRF-(12-41), or the selective nonpeptide CRF1 receptor antagonist, NBI 27914. Finally, in cultured rat pituitary cells that express native CR F1 receptors, CRF-SAP suppressed CRF-induced(1 nM) ACTH release. GnRH (1-10 nM) stimulated LH release was also assessed in the same pituitary cultures . Although there was a slight decrease in LH release from these cultures, t his decrease was observed with CRF-SAP or SAP alone, suggesting that the re sponse was nonspecific. Taken together, these results suggest the utility o f CRF-SAP as a specific and subtype-selective tool for long term impairment of CRF1 receptor-expressing cells.