title: Selective impairment of corticotropin-releasing factor1 (CRF1) receptor-mediated function using CRF coupled to saporin.
abstract: 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 CRF2alpha 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.
authors.affiliation: Neurocrine Biosciences, Inc., San Diego, California 92121, USA. dmaciejewski@neurocrine.com
authors.forename: D
authors.initials: D
authors.lastname: Maciejewski-Lenoir
authors.forename: S C
authors.initials: SC
authors.lastname: Heinrichs
authors.forename: X J
authors.initials: XJ
authors.lastname: Liu
authors.forename: N
authors.initials: N
authors.lastname: Ling
authors.forename: A
authors.initials: A
authors.lastname: Tucker
authors.forename: Q
authors.initials: Q
authors.lastname: Xie
authors.forename: D A
authors.initials: DA
authors.lastname: Lappi
authors.forename: D E
authors.initials: DE
authors.lastname: Grigoriadis
mesh_terms: D000324:Adrenocorticotropic Hormone / Q000378:metabolism; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D003346:Corticotropin-Releasing Hormone / Q000494:pharmacology*; D005260:Female; D007987:Gonadotropin-Releasing Hormone / Q000494:pharmacology; D006801:Humans; D000922:Immunotoxins / Q000494:pharmacology; D007739:L Cells; D007986:Luteinizing Hormone / Q000378:metabolism; D051379:Mice; D009699:N-Glycosyl Hydrolases; D010902:Pituitary Gland / Q000166:cytology / Q000187:drug effects / Q000502:physiology; D010940:Plant Proteins / Q000494:pharmacology*; D051381:Rats; D018019:Receptors, Corticotropin-Releasing Hormone / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D011994:Recombinant Proteins / Q000187:drug effects / Q000378:metabolism; D054789:Ribosome Inactivating Proteins, Type 1; D000076985:Saporins; D014162:Transfection; D000099052:CRF Receptor, Type 1
chemical_list: D002352:Carrier Proteins; D000922:Immunotoxins; D010940:Plant Proteins; D018019:Receptors, Corticotropin-Releasing Hormone; D011994:Recombinant Proteins; D054789:Ribosome Inactivating Proteins, Type 1; C067133:corticotropin releasing factor-binding protein; D007987:Gonadotropin-Releasing Hormone; D000099052:CRF Receptor, Type 1; D000324:Adrenocorticotropic Hormone; D007986:Luteinizing Hormone; D003346:Corticotropin-Releasing Hormone; D009699:N-Glycosyl Hydrolases; D000076985:Saporins