Approaches in the study of the posttranslational processing of opioid peptide peptide precursors: Focus on prodynorphin

77 APPROACHES IN THE STUDY OF THE POSTTRANSLATIONAL PROCESSING OF OPIOID PEP'rIDE PEPTIDE PRECURSORS: FOCUS ON PRODYNORPHIN Day R, Vieau D, Seidah NG, Clinical Research Institute of Montreal, Montreal, Quebec, Canada. The opioid peptide precursor prodynorphin (proDyn) is differentially processed depending on the tissues in which it is expressed. These tissues also express different combinations of prohormone convertases (PCs) which are known to process precursor proteins at paired (KR, RR, RK or KK) or single (R) basic amino acid residues (K=lysine and R=arginine), thereby suggesting a rationale for tissue-specific processing. A comparison of proDyn processing in various tissues with PC1 and PC2 expression levels suggests that PC1 is responsible for the production of high molecular weight (HMWO intermediates of proDyn while PC2 is important for the further processing of proDyn to the smaller opioid peptides such as dynorphin A 1-17 (Dyn A17) or dynorphin B 1-13 (Dyn B13). These data are supported by vaccinia virus overexpression experiments of PC1 or PC2 with proDyn in different mammalian cell lines. Other PCs may be involved in proDyn processing in tissues lacking PC1 or PC2 expression. In the adrenal cortex and testicular Sertoli cells, a strong correlation exists between the expression of proDyn and PC5. Finally, we report that the mouse pancreatic 13TC3cell line, known for its expression of proinsulin, endogenously produces high levels of proDyn mRNA and proDyn derived peptides. The 13TC3 expresses high levels of both PC1 and PC2 but not PC5. This cell line will be a useful model to study the ordered cleavage events of proDyn processing but may also be important in a re-evaluating proDyn's role in pancreatic funclior~ Male Sprague Dawley rats (250-300 g) were used. The analysis of proDyn derived peptides using Sephadex G-50 and HPLC combined Dyn A17, Dyn B13 and ct-neo-endorphin (oNE) RIAs has been previously described (1). PCs and proDyn mRNAs were detected by Northern blot and in situ hybridization as previously described (2, 3). ~TC3 cells were obtained from Dr. D. Hanahan (4). The opioid peptides Dyn A17, Dyn B13 and ctNE are processed from proDyn. Each peptide is flanked by pairs of basic residues (KR) or a single basic residue (R). ProDyn contains 4 KR sites but also one R site critical for Dyn B13 production. The enzymes which cleave precursors at either pairs of basic and/or single basic amino acids have been characterized and are known as the PCs and include PC1, PC2, furin, PC4, PACE4, and PC5 (5, 6). The PCs are expressed in a tissue specific manner in the CNS and periphery (6). ProDyn is also expressed in various tissues including the CNS (striatum, hypothalamus, cortex, spinal cord), pituitary (anterior lobe gonadotrophs and intermediate lobe melanotrophs), adrenal cortex (zona fasiculata and reticularis), and testicular Sertoli cells. ProDyn is also differentially processed to HMW intermediates (> 4 KDa) or to the smaller opioid peptides (< 4 KDa). In this study we compared the steady state levels of proDyn derived peptides (HMW intermediates and smaller opioid peptides) in different tissues and established a correlation with the expression of PC1 and PC2 (Table I). Table I. Tissue specific processing of proDyn: correlation with PC1 and PC2 mRNA levels. PC1:PC2

Tissue

HMW Intermediates

Anterior pituitary Spinal cord Hypothalamus Striatum

+++++ +++ + ±

(mRNA ratio) PC1 >> PC2 PC1 > PC2 PC1 > PC2 PC1 << PC2

Er~ products: Dyn AIT,I~n B13 + ++ ++++ +++++

The anterior lobe gonadotrophs process ProDyn principally into 10 and 8 KDa HMW intermediate forms (1). Using in situ hybridization, we observed high levels of PC1 but not PC2 mRNA in gonadotrophs. In contrast, the striatum contains only completely processed forms of proDyn such as Dyn A17 or Dyn B13. Both Northern blot and in situ hybridization analysis reveal high levels of PC2 but much lower levels of PC1 mRNA expression in the striatum. Similarly, we compared other proDyn expressing tissues (spinal cord and hypothalamus) for PC1 and PC2 expression (Table I). We note that as the PCI:PC2 ratio shifts in favor of PC2, so does the degree of proDyn processing from HMW intermediates to end products. In support of this correlation, using the vaccinia virus

78 overexpression system, we noted that PC1 and proDyn co-expression resulted in the formation of the 8 and 10 KDa HMW intermediates (7). Further proDyn processing required the action of PC2. ProDyn is also expressed in tissues lacking PC1 and PC2 expression. Two examples are the adrenocortical cells (Fig 1B) and testicular Sertoli cells (Fig 1F). Neither cells express PC1 or PC2, yet proDyn processing is observed (8, 9), suggesting the involvement of other enzymes. High levels of PC5 are observed in these cells (Fig 1A and Fig 1E), suggesting a role for PC5 in proDyn processing. While PC5 may have a specific role in the processing of proDyn, it may also be acting in concert with PC1 to yield a defined set of products. This could be the case in the anterior lobe where PC5 and proDyn mRNA distributions are correlated (Fig 1C and 1D), suggesting that gonadotrophs express PC5. We are currently investigating PC5's cleavage specificity on proDyn using vaccinia virus expression. Posttranslational processing studies are simplified by the availability of cell lines expressing high levels of the precursor of interest. To date cell lines expressing very high levels of proDyn have not been reported. We examined over 24 mammalian cell lines for proDyn mRNA. The mouse insulinoma 13TC3 was the only cell line which expressed very high proDyn mRNA levels. I~TC3cells produce insulin but expression of other peptides has not been reported. Our analysis of proDyn peptides in I~TC3 cells reveals the presence of opioid peptides (aNE and Dyn B13) but no HMW intermediates, indicating a high processing activity in these cells. In order to gain some understanding of how proDyn is processed in the ~3TC3, we examined PC mRNA levels in these cells. 13TC3 cells express high levels of PC1 and PC2 but not PC5 mRNA. As with the observed tissue correlation (Table I), high PC2 mRNA levels in I~TC3 cells may be important in processing proDyn to opioid peptides. We conclude that various PCs are responsible for proDyn processing, however each PC acts at specific sites to yield different Fig 1. In situ hybridization products. The relative levels of PCs co-expressed in specific cells will of PC5 and proDyn mRNA define the products obtained. Further studies are needed to in the (A, B) adrenal, (C, D) investigate the action of other PCs (PACE4 and furin) on proDyn pituitary and (E, F) testis. Abbreviations: cx=cortex; processing. The observation of proDyn expression in ~TC3 cells m=medulla; AP=anterior should be invaluable in defining temporal events in proDyn pituitary; IL=intermediate processing. Since 13TC3 cells are derived from insulin producing lobe; NL=neural lobe. pancreatic beta cells, it may be of interest to re-evaluate the involvement of proDyn peptides in pancreatic function. Furthermore it will be of interest to determine what factors are responsible for the selective expression of proDyn in I~TC3 cells. This work was supported by the Medical Research Council of Canada (MTl1268). R.D. is a scholar of the Fonds de la Recherche en Sant~ du Qu(~bec. REFERENCES 1. R. Day and H. Akil (1989) Endocrinology 124, 2392-2405 2. R. Day, M.K.-H. SchMer, S.J. Watson, M. Chr6tien and N.G. Seidah (1992) Molecular Endocrinology 6, 1559-1569 3. R. Day, M.K.-H. Sch~fer, M.W. Collard, E. Weihe, and H. Akil (1993) Endocrinology 133, 26522659 4. S. Efrat, S. Linde, H. Kofod, D. Spector, M. Delannoy, S. Grant, D. Hanahan, and S. Baekkeskov (1988) Proceedings of the National Academy of Sciences USA 85, 9037-9041 5. N.G. Seidah, R. Day, M. Marcinkiewicz, S. Benjannet and M. Chr~fien (1991) Enzyme 45, 271-284 6. N.G. Seidah, M. Chr6tien and R. Day (1994) Biochimie 76, (in press) 7. A. Dupuy, I. Lindberg, Y. Zhou, H. Akil, C. Lazure, M. Chr~tien, N.G. Seidah and R. Day (1994) FEBS Letters 337, 60-65 8. M.W. Collard, R. Day, H. Akil, M.D. Uhler and J.O. Douglas (1990) Molecular Endocrinology 4, 1488-1496 9. R. Day, M.K.-H. Sch~ifer, M.W. Collard, S.J. Watson and H. Akil (1991) Proceedings of the National Academy of Sciences USA 88, 1320-1324