GENERATION AND CHARACTERIZATION OF AN ANTISERUM REACTIVE WITH A PROTEOLYTIC PROCESSING SITE WITHIN RAT PROCORTICOTROPHIN-RELEASING HORMONE

In this paper we report the generation of an antibody specific for the cleavage site within procorticotrophin-releasing hormone (proCRH) at the N-terminus proCRH/CRH (1-41) junction. Using radioimmunoassay tech niques we show that the antibody generated (781) cross-reacts specific ally with the proCRH (137-150) Tyr fragment, corresponding to the clea vage site within the full length precursor molecule. The anti-cleavage site antibody does not crossreact with the endoproteolytic products o riginated from the CRH precursor molecule, i.e. CRH (1-41) or proCRH ( 125-151) or with any of the CRH-immunoreactive fragments tested i.e. C RH (36-41), CRH (1-20) and CRH (30-41). It also shows no cross-reactiv ity with CRH-related substances from other species, i.e. urotensin I ( fish) and sauvagine (frog). The cleavage site antibody (781), recogniz es the full length proCRH molecule in Western blotting and in liquid p hase radioimmunoassay from transfected CHO-K1 cells expressing the ful l length pre-proCRH cDNA. Using immunofluorescence and immunoprecipita tion techniques followed by SDS-PAGE and autoradiography, we confirm t he presence of the intact CRH precursor molecule within the nucleus an d the cytoplasm of stably transfected CHO-K1 cells expressing immunore active proCRH. The immunofluorescence studies using primary cultures o f hypothalamic neurons, show that immunoreactive (IR) proCRH is locali zed within the perinuclear region and was also seen along the neuronal processes where it accumulates at their tips. Our results, therefore, show that this antibody will be an invaluable tool in the study of in tracellular trafficking in relation to the endoproteolytic processing of the CRH precursor molecule.