Selective impairment of corticotropin-releasing factor1 (CRF1) receptor-mediated function using CRF coupled to saporin.
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.
Maciejewski-Lenoir
D
D
Neurocrine Biosciences, Inc., San Diego, California 92121, USA. dmaciejewski@neurocrine.com
Heinrichs
S C
SC
Liu
X J
XJ
Ling
N
N
Tucker
A
A
Xie
Q
Q
Lappi
D A
DA
Grigoriadis
D E
DE
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
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