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Selective Impairment of Corticotropin-Releasing Factor₁ (CRF₁) Receptor-Mediated Function Using CRF Coupled to Saporin¹

Neurocrine Biosciences, Inc. (D.M.-L.), San Diego, California 92121; Department of Psychology, Boston College (S.C.H.), Chestnut Hill, Massachusetts 02467; and Advanced Targeting Systems (D.A.L.), San Diego, California 92121

Address all correspondence and requests for reprints to: D. Maciejewski-Lenoir, Ph.D., Neurocrine Biosciences, Inc., 10555 Science Center Drive, San Diego, California 92121. E-mail: dmaciejewski{at}neurocrine.com

CRF is the main component in the brain neuropeptide effector system responsible for the behavioral, endocrine, and physiological activation that accompanies 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 ribosome-inactivating protein, saporin (CRF-SAP), for the purpose of targeted inactivation of CRF receptor-expressing cells. Cytotoxicity measurements revealed that CRF-SAP (1–100 nM) produced concentration-dependent and progressive cell death over time in CRF₁ receptor-transfected L cells, but at similar concentrations had no effect on CRF₂ receptor-transfected cells. The CRF-SAP-induced toxicity in CRF₁-transfected cells was prevented by coincubation with the competitive CRF₁/CRF₂ receptor peptide antagonist, [D-Phe¹²]CRF-(12–41), or the selective nonpeptide CRF₁ receptor antagonist, NBI 27914. Finally, in cultured rat pituitary cells that express native CRF₁ 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, this decrease was observed with CRF-SAP or SAP alone, suggesting that the response was nonspecific. Taken together, these results suggest the utility of CRF-SAP as a specific and subtype-selective tool for long term impairment of CRF₁ receptor-expressing cells.

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