Procorticotrophin releasing hormone is endoproteolytically processed by the prohormone convertase PC2 but not by PC1 within stably transfected CHO-K1 cells.

Hormones and neuropeptides are generally synthesized as large precursor molecules: i.e. prohormones and proneuropeptides which undergo endoproteolysis to produce biologically active peptides. Corticotrophin releasing hormone (CRH) is a 41 aminoacid peptide which plays a major role in the regulation of the endocrine response to stress. CRH( 1-41) is generated after endoproteolytic cleavage at pairs of basic aminoacid residues from its precursor molecule: proCRH [ I ] . Two enzymes which belong to the subtilisin family of serine proteases, PC I (also called PC3) and PC2 might be the candidates for the endoproteolytic processing of proCRH, due to their cleavage specificities and their expression in tissues of endocrine-neuroendocrine origin. In order to investigate if PCI or PC2 are capable of correctly cleaving proCRH within CHO-KI cells we transfected an expression plasmid containing the PC I cDNA and PC2 cDNA into stably transfected CHO-KI cells already expressing proCRH (clone 1/22) [2]. Expression vectors carrying PCI or PC2 cDNAs were constructed by ligating the 2,615 bp and 2,223 bp fragments of PCI and PC2 cDNAs respectively, into the PEE6hCMVne eukaryotic expression vector under the control of the human cytomegalovirus promoter. This vector encodes for the antibiotic resistance gene neomycin as selectable marker. Transfection was carried out using the calcium phosphate precipitation method and selection of resistant cells was achieved using G418 sulphate at 900 p g / d in the grow media. Clones were picked up at random and screened for the expression of PCI and PC2 enzymes by immunofluorescence using specific antibodies to PC 1 and PC2 (kindly provided by Dr. I. Lindberg, Lousiana State University, USA). Colocalization of immunoreactive (1R)-CRH and PCI or PC2 was studied by double immunofluorescence. Cells were grown to 70-80 % confluency on poly-L-lysine coated coverslips in 6 well tissue culture plates. They were washed in phosphate buffered saline (PBS) before fixing in 4 % paraformaldehyde, 0.12 M sucrose in PBS for 20 minutes. After several washes in PBS the cells were permeabilised in 0.5 % Triton X-100 in PBS for 5 minutes, followed by several washes in PBS. They were then incubated in 10 % (V/V) normal horse serum (NHS) in PBS for 1 hour and then I % ( V N ) NHS in PBS for 10 minutes to block nonspecific binding. Anti-CRH (sheep, 1/200 dilution) and antiPCI or PC2 antibodies (rabbit, M O O dilution) were added together in 1 % ( V N ) NHS in PBS. The cells were incubated in the buffer containing the primary antibodies for 1 hour at room temperature, then washed several times in PBS and incubated for 1 h at room temperature in the appropiate dilution of the secondary antibodies in 1 9% NHS in PBS. The secondary antibodies employed were goat anti-rabbit conjugated to rhodamine (1150 dilution) and donkey anti-sheep conjugated to fluorescein (11100 dilution). After 3 further washes in PBS, the coverslips were mounted in 2.5 % propyl gallate in 85 % glycerol, and the edges sealed using nail varnish. The immunoreactive (IR) staining pattern of CRH showed a reticular cytoplasmic distribution as well as nuclear foci within both stably transfected CHO-KI cells co-expressing CRH and PCI (1/22/pC1 clone) (Fig. 1) or CRH and PC2 (MT-PC2ICRH clone) (Fig 2). Double labelling immunofluorescence studies using Figure 2 Co-localiuuon of IR CRH and IR FC2 within robly lrasicclcd CHO KI cell, IMT PCUCRH) Pur l A IR-CRH m MT FCUCRH cclls pawl B IR FC2 m t h r a m cells shown m p m l A Phi= conU.11 mcwaph of a l l r shown m panel A panel D nucla o i cclls rhown m panel A u i w a l u ~ d wing Ihc n u c l w hmahcmcd d e r 4 6diarmnc-2 pknylindole (DAPI) Scale bu= 25 prn