0013-7227/91/1296-3274$03.00/0 Endocrinology Copyright © 1991 by The Endocrine Society
Vol. 129, No. 6 Printed in U.S.A.
Biosynthesis of Human Growth Hormone-Releasing Hormone (hGRH) in the Pituitary of hGRH Transgenic Mice* ANOOP K. BRAR, THOMAS R. DOWNS, EDGAR P. HEIMER, ARTHUR M. FELIX, AND LAWRENCE A. FROHMAN Division of Endocrinology and Metabolism (A.K.B., T.R.D., L.A.F.), Department of Internal Medicine, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267; and Peptide Research Department (E.P.H., A.M.F.), Roche Research Center, Hoffmann-LaRoche, Inc., Nutley, New Jersey 07110
hGRH(l-40)-OH were present, and very little intracellular 35Spro-hGRH remained. A progressive decrease in the ratio of immunoprecipitable pro-hGRH to mature hGRH peptides and an increase in the ratio of hGRH(l-40)-OH to hGRH(l-44)NH2 was observed in the two chase periods. In medium, [35S] hGRH(l-44)-NH2 was detectable at all times, whereas only minimal amounts of [35S]hGRH(l-40)-OH were present. Labeled and unlabeled pro-hGRH in cell extracts was also detected with anti-hGCTP serum, and another peak, which coeluted with synthetic hGCTP, was also identified. The low molar ratio of intracellular immunoreactive hGCTP to hGRH («. |
10
..••"•'
3277
10 "
200
O 100 10
20
30
40
50
60
70
80
The fractions from the 4 h pulse incubation study shown in Fig. 1 were assayed for hGCTP and immunoprecipitated with anti-hGCTP serum to determine [35S] hGCTP levels. On the basis of preliminary HPLC experiments, only the last 30 fractions of the chromatography of pituitary cell extracts were measured (Fig. 4). The major immunoreactive peak coeluted with pro-hGRH. Several other immunoreactive peaks were also identified, one of which had a retention time identical to that of synthetic hGCTP. The major peak of hGCTP immuno2. Time-dependent changes in intracellular [35S]hGRH precursor-product ratios
TABLE
30
40
50
FRACTION
FiG. 2. HPLC elution profiles of hGRH-IR and immunoprecipitable [MS]hGRH in pituitary cells after a 0.5-h pulse (top panel), a 0.5-h pulse plus a 0.5-h chase (middle panel), and a 0.5-h pulse plus a 4-h chase (lower panel). Details are as in Fig. 1.
hGRH Form
0.5-h Pulse
0.5-h Chase
4-h Chase
Pro-hGRH hGRH-44 + hGRH-40
3.23
0.82
0.34
hGRH-40 hGRH-44
0.10
0.17
1.10
The values represent the ratios of immunoprecipitable radioactivity of the individual hormone forms present in the cell extracts, as shown in Fig. 2.
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3278
BIOSYNTHESIS OF HUMAN GRH
Endo-1991 Vol 129 • No 6
radioactivity precipitated by the hGCTP antibody, one of which coeluted with the hGCTP standard.
Discussion
- .2
30
20
40
50
60
70
80
FRACTION
FIG. 3. HPLC elution profiles of hGRH-IR and immunoprecipitable [35S]hGRH in incubation media after a 0.5-h pulse {top panel), a 0.5-h pulse plus a 0.5-h chase (middle panel), and a 0.5-h pulse plus a 4-h chase (lower panel). Details are as in Fig. 1. 2000
1.0
4 h Pulse CELLS 1500
.75
I50XI
1000
(27X) .25
500
10
20
30
40
50
60
70
80
FRACTION
FlG. 4. HPLC elution profiles of hGCTP-IR and immunoprecipitable [35S]hGCTP in pituitary cells after a 4-h pulse. Details are as in Fig. 1.
precipitable radioactivity was present in pro-hGRH. Several of the other immunoreactive peaks also contained
The present data provide evidence for the biosynthesis of hGRH in pituitaries of transgenic mice in vitro. The results have shown the incorporation of a radiolabeled amino acid (methionine) into the hGRH precursor and its processing to the mature forms of the hormone. Demonstration of this process was facilitated by the high levels (100-500 times that in rat hypothalamus) and high rate of hGRH synthesis from a transgene that is constitutively expressed in this tissue. Although the pituitary is not the normal site of GRH gene expression, it contains the necessary complement of processing enzymes to convert the prohormone to the mature hormonal peptide forms (8). The fact that the efficiency of prohormone processing is not as great as in the hypothalamus aided the present studies since it permitted greater accumulation of the prohormone and facilitated demonstration of the processing pathway. The pulse-chase study clarified several aspects of hGRH biosynthesis that had previously been assumed and/or open to speculation. First, the peak tentatively identified as the hGRH precursor (pro-hGRH) in previous studies on the basis of its molecular size and relative immunoreactivity with two separate hGRH RIAs (8) has been shown to accumulate [35S]methionine during a pulse period and to have this radiolabel diminish during a chase period. Furthermore, this peak could be demonstrated with an antibody to hGCTP as well as to hGRH. Second, the temporal sequence of accumulation of radioactivity into the two mature forms of hGRH, hGRH(l-44)-NH 2 and hGRH(l-40)-OH, indicates that the former serves as a precursor for the latter. Incorporation of radioactive label was detected in hGRH(l-44)NH2 in both cells and media by the end of the pulse period but did not appear in hGRH(l-40)-OH until the end of the first chase period. The present results provide evidence for the incorporation of radioactivity into several peaks that exhibit hGCTP immunoreactivity, one of which coelutes with pro-hGRH. The higher level of radioactivity precipitated by the hGCTP antibody as compared to the hGRH antibody may reflect different affinities of the two antibodies for pro-hGRH and/or other C terminally extended molecules. However, only a small amount of immunoprecipitable radioactivity coeluted with the synthetic hGCTP standard. These results, together with the low intracellular level of hGCTP-IR [less than 2% of hGRH(l-44)-NH 2 and hGRH(l-40)-OH on a molar basis] and the absence of detectable levels of hGCTP-IR in the incubation media (data not shown), suggest that
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BIOSYNTHESIS OF HUMAN GRH
hGCTP, once synthesized, is rapidly degraded. The present results suggest the proposed schema of hGRH biosynthesis shown in Fig. 5. This extends the previously proposed pathways, based on the amino acid sequences of hGRH and the hGRH precursor as deduced from its cDNA (1, 2). After removal of the signal peptide, processing of pro-hGRH is initiated first by cleavage at positions 46-47 and then 45-46 by a trypsin-like endopeptidase and a carboxypeptidase, resulting in hGRH(l45)-OH and hGCTP formation. An amide group is then transferred from Gly45 to Leu44 by peptidyl glycine aamidating monooxygenase (PAM) (17). This transamidating step has recently been shown to occur using synthetic hGRH(l-45) and PAM (18, 19). Based on immunohistochemical studies using antisera specific for the carboxyl terminus of hGCTP it is likely that hGCTP also serves as a substrate for PAM, resulting in an amide transfer from Gly77 to Gin76 (5). The present study cannot resolve this possibility, since these two compounds would not be expected to be separated under the conditions employed for HPLC (8). Finally, peptidases cleave at positions 41-42 and 40-41 of hGRH(l-44)-NH 2 to generate hGRH(l-40)-OH. An obligatory role of hGRH(l-44)-NH 2 in the generation of hGRH(l-40)-OH is also suggested by the absence of both hormonal forms in the liver of transgenic hGRH mice, where there is accumulation of hGRH(l-45)-OH (8) as a consequence of the absence of PAM gene expression in this tissue (17). Some hGRH-secreting tumors also have been prcpro-GRH -30
- 1 0
Met
Arg-Arg-Tyr.
pro-GRH
1
I
40
41
44
45
46 47
77
Ala-Arg....Leu-Gly-Arg-Gln
Gly
Signal peptide cleavage
1
40
Tyr.
Ala-Arg....Leu-Gly-Arg-Gln
41
44
45
46
47
Jl
77
Gly
Endopeptidase + Carboxypeptidase
GRH(l-45)-OH
GCTP
1
40
41
44
45
Tyr.
Ala-Arg....Leu-Gly
47
77
Gin
Glv
Peptidylglycine a-amidating monooxygenase (PAM)
GRH(l-44)-NH 2 1
40
Tyr.
Ala-Arg....Leu-NH2
41
IJ
44
Endopeptidase + Carboxypeptidase
GRH(1-40)-OH 1
40
Tyr.
Ala
FIG. 5. Proposed schema for hGRH biosynthesis.
3279
shown to contain hGRH(l-37)-OH (13, 20). The present results do not clarify whether this peptide is derived by further modification of hGRH(l-40)-OH or directly from hGRH(l-44)-NH2. In the present studies there was no evidence for hGRH(l-37)-OH, which would have been expected to elute slightly beyond that of hGRH (1-40)OH. The transgenic pituitary cells and media contained several other GRH-IR peaks in addition to those that could be identified. Two major peaks, both of which incorporated considerable radioactive label, eluted just ahead of hGRH(l-44)-NH2. Neither of these peaks was recognized by the hGCTP antiserum (not shown). Their positions are consistent with those of hGRH(3-44)-NH2 and hGRH(3-40)-OH, the primary hGRH metabolites observed in plasma (21, 22). The enzyme responsible for this metabolic conversion, dipeptidylpeptidase, type IV, is not detectable in the pituitary (our unpublished observations), though this tissue does contain aminopeptidases that could possibly generate these compounds. These immunoreactive peaks have previously been noted in pituitary and, at relatively lower concentrations, in other tissues from transgenic hGRH mice (8). However, the small quantity of each of these peptides present in the pituitary precludes their absolute identification at the present time. Finally, it is of interest to note that only in the human species have two mature forms of GRH been identified. Although hGRH(l-40)-OH exhibits a longer half-life than does hGRH(l-44)-NH 2 (21), the two peptides are biologically equipotent and the physiological importance of there being two releasing hormone forms in humans, if any, is unknown. Both mouse and rat GRH contain an Arg41 (23, 24), as does hGRH, and both the mouse hypothalamus and pituitary (8, present study) contain the necessary converting enzyme(s). Since neither mouse nor rat GRH is carboxyl-terminally amidated, it is tempting to speculate that carboxyl-terminal amidation is a substrate prerequisite. However, porcine GRH, which both contains Arg41 and is carboxyl-terminally amidated (25), exists only in a 44-amino acid form. Endopeptidase cleavage at monobasic sites is believed to require specific neighboring amino acid sequences (11). The difference in carboxyl-terminal sequences of the other mammalian GRHs and hGRH (23, 26) may explain the lack of their further processing. In summary, the present pulse-chase studies have demonstrated the processing of the hGRH precursor to both mature forms of hGRH and have provided evidence that hGRH(l-40)-OH is derived from hGRH(l-44)-NH 2. It should be emphasized that the present pulse-chase studies were performed in pituitary rather than hypothalamus. Although mouse hypothalamus is capable of generating hGRH(l-40)-OH (8), the possibility cannot be
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 03 August 2016. at 18:49 For personal use only. No other uses without permission. . All rights reserved.
BIOSYNTHESIS OF HUMAN GRH
3280
excluded that the processing mechanism in this tissue varies from that in the pituitary. Nevertheless, the fact that these studies were conducted in a different species as well as tissue suggests that the processing enzymes required for human GRH biosynthesis are present in multiple tissues and functionally preserved in at least two mammalian species.
Acknowledgments We thank Jane Withrow for her excellent technical assistance.
References 1. Gubler U, Monahan JJ, Lomedico PT, Bhatt RS, Collier KJ, Hoffman BJ, Bohlen P, Esch F, Ling N, Zeytin F, Brazeau P, Gage LP 1983 Cloning and sequence analysis of cDNA for the precursor of human growth hormone-releasing factor, somatocrinin. Proc Natl Acad Sci USA 80:4311-4314 2. Mayo KE, Vale W, Rivier J, Rosenfeld M-G, Evans RM 1983 Expression-cloning and sequence of cDNA encoding human growth hormone-releasing factor. Nature 306:86-88 3. Ling N, Esch F, Bohlen P, Wehrenberg WB, Guillemin R 1984 Isolation, primary structure and synthesis of human hypothalamic somatocrinin: growth hormone-releasing factor. Proc Natl Acad Sci USA 81:4302-4306 4. Bohlen P, Brazeau P, Bloch B, Ling N, Gaillard R, Guillemin R 1983 Human hypothalamic growth hormone releasing factor (GRF): evidence for two forms identical to tumor derived GRF-44NH2 and GRF-40. Biochem Biophys Res Commun 114:930-936 5. Bloch B, Baird A, Ling N, Guillemin R 1986 Immunohistochemical evidence that growth hormone-releasing factor (GRF) neurons contain an amidated peptide derived from cleavage of the carboxylterminal end of the GRF precursor. Endocrinology 118:156-162 6. Hammer RE, Brinster RL, Rosenfeld MG, Evans RE, Mayo KE 1985 Expression of human growth hormone-releasing factor in transgenic mice results in increased somatic growth. Nature 315:413-416 7. Brar A, Brinster R, Frohman LA 1989 Immunohistochemical analysis of human growth hormone-releasing hormone gene expression in transgenic mice. Endocrinology 125:801-809 8. Frohman LA, Downs TR, Kashio Y, Brinster R 1990 Tissue distribution and molecular heterogeneity of human growth hormone-releasing factor in the transgenic mice. Endocrinology 127:2149-2156 9. Low MJ, Hammer RG, Goodman RH, Habener JF, Palmiter RD, Brinster RL 1985 Tissue-specific post-translational processing of pre-prosomatostatin encoded by a metallothionein-somatostatin fusion gene in transgenic mice. Cell 41:211-219 10. Fisher JM, Scheller RH 1988 Prohormone processing and the secretory pathway. J Biol Chem 263:16515-16518
Endo • 1991 Voll29«No6
11. Devi L 1991 Consensus sequence for processing of peptide precursors at monobasic sites. FEBS Lett 280:189-194 12. Sambrook J, Fritsch EE, Maniatis T 1989 Molecular Cloning, A Laboratory Manual, ed 2. Cold Spring Harbor Laboratory, Cold Spring Harbor 13. Frohman LA, Downs TR 1986 Measurement of growth hormonereleasing factor. Methods Enzymol 124:371-389 14. Brar AK, Coleman TA, Kopchick JJ, Frohman LA 1990 Expression of a cytomegalovirus-human growth hormone-releasing hormone precursor fusion gene in transfected GH3 cells. Mol Cell Endocrinol 71:105-115 15. Barany G, Merrifield RB 1980 Solid phase peptide synthesis. In: Gross E, Meinhofer J (eds) The Peptides: Analysis, Synthesis, Biology. Academic Press, New York, vol 2:1-284 16. Green N, Alexander H, Olson A, Alexander S, Shinnick TM, Sutcliffe JG, Lerner RA 1982 Immunogenic structure of the influenza virus hemagglutinin. Cell 28:477-487 17. Eipper BA, Park LP, Dickerson IM, Keutmann MI, Thiele EA, Rodriguez H, Schofield PR, Mains RE 1987 Structure of the precursor to an enzyme mediating COOH-terminal amidation in peptide biosynthesis. Mol Endocrinol 1:777-790 18. Engels JW, Glauder J, Mullner H, Tripier D, Uhlmann E, Wetekam W 1987 Enzymatic amidation of recombinant (Leu27) growth hormone releasing hormone-Gly45. Protein Engineering 1:195-199 19. Bongers J, Heimer EP, Campbell RM, Felix AM, Merkler DJ, aAmidating enzyme catalyzed synthesis of peptide-amides from glycine-extended precursors: human growth hormone releasing factor and analogs as examples. In: Rivier J, Smith J (eds) Proceedings of the 12th American Peptide Symposium. ESCOM, Boston, in press 20. Guillemin R, Brazeau P, Bohlen P, Esch F, Ling N, Wehrenberg WB 1982 Growth hormone-releasing factor from a human pancreatic tumor that caused acromegaly. Science 218:585-587 21. Frohman LA, Downs TR, Williams TC, Heimer EP, Pan Y-CE, Felix AM 1986 Rapid enzymatic degradation of growth hormonereleasing hormone by plasma in vitro and in vivo to a biologically inactive product cleaved at the NH2 terminus. J Clin Invest 78:906913 22. Frohman LA, Downs TR, Heimer EP, Felix AM 1989 Dipeptidylpeptidase IV and trypsin-like enzymatic degradation of human growth hormone-releasing hormone in plasma. J Clin Invest 83:1533-1540 23. Frohman MA, Downs TR, Chomczynski P, Frohman LA 1989 Cloning and characterization of mouse growth hormone-releasing hormone (GRH) cDNA: increased GRH mRNA levels in the growth hormone deficient lit/lit mouse. Mol Endocrinol 3:15291536 24. Spiess J, Rivier J, Vale W 1983 Characterization of rat hypothalamic growth hormone-releasing factor. Nature 303:532-535 25. Bohlen P, Esch F, Brazeau P, Ling N, Guillemin R 1983 Isolation and characterization of the porcine growth hormone releasing factor. Biochem Biophys Res Commun 116:726-734 26. Frohman LA, Jansson J-0 1986 Growth hormone-releasing hormone. Endocr Rev 7:223-253
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Vol. 129, No. 6 Printed in U.S.A.
Biosynthesis of Human Growth Hormone-Releasing Hormone (hGRH) in the Pituitary of hGRH Transgenic Mice* ANOOP K. BRAR, THOMAS R. DOWNS, EDGAR P. HEIMER, ARTHUR M. FELIX, AND LAWRENCE A. FROHMAN Division of Endocrinology and Metabolism (A.K.B., T.R.D., L.A.F.), Department of Internal Medicine, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267; and Peptide Research Department (E.P.H., A.M.F.), Roche Research Center, Hoffmann-LaRoche, Inc., Nutley, New Jersey 07110
hGRH(l-40)-OH were present, and very little intracellular 35Spro-hGRH remained. A progressive decrease in the ratio of immunoprecipitable pro-hGRH to mature hGRH peptides and an increase in the ratio of hGRH(l-40)-OH to hGRH(l-44)NH2 was observed in the two chase periods. In medium, [35S] hGRH(l-44)-NH2 was detectable at all times, whereas only minimal amounts of [35S]hGRH(l-40)-OH were present. Labeled and unlabeled pro-hGRH in cell extracts was also detected with anti-hGCTP serum, and another peak, which coeluted with synthetic hGCTP, was also identified. The low molar ratio of intracellular immunoreactive hGCTP to hGRH («. |
10
..••"•'
3277
10 "
200
O 100 10
20
30
40
50
60
70
80
The fractions from the 4 h pulse incubation study shown in Fig. 1 were assayed for hGCTP and immunoprecipitated with anti-hGCTP serum to determine [35S] hGCTP levels. On the basis of preliminary HPLC experiments, only the last 30 fractions of the chromatography of pituitary cell extracts were measured (Fig. 4). The major immunoreactive peak coeluted with pro-hGRH. Several other immunoreactive peaks were also identified, one of which had a retention time identical to that of synthetic hGCTP. The major peak of hGCTP immuno2. Time-dependent changes in intracellular [35S]hGRH precursor-product ratios
TABLE
30
40
50
FRACTION
FiG. 2. HPLC elution profiles of hGRH-IR and immunoprecipitable [MS]hGRH in pituitary cells after a 0.5-h pulse (top panel), a 0.5-h pulse plus a 0.5-h chase (middle panel), and a 0.5-h pulse plus a 4-h chase (lower panel). Details are as in Fig. 1.
hGRH Form
0.5-h Pulse
0.5-h Chase
4-h Chase
Pro-hGRH hGRH-44 + hGRH-40
3.23
0.82
0.34
hGRH-40 hGRH-44
0.10
0.17
1.10
The values represent the ratios of immunoprecipitable radioactivity of the individual hormone forms present in the cell extracts, as shown in Fig. 2.
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3278
BIOSYNTHESIS OF HUMAN GRH
Endo-1991 Vol 129 • No 6
radioactivity precipitated by the hGCTP antibody, one of which coeluted with the hGCTP standard.
Discussion
- .2
30
20
40
50
60
70
80
FRACTION
FIG. 3. HPLC elution profiles of hGRH-IR and immunoprecipitable [35S]hGRH in incubation media after a 0.5-h pulse {top panel), a 0.5-h pulse plus a 0.5-h chase (middle panel), and a 0.5-h pulse plus a 4-h chase (lower panel). Details are as in Fig. 1. 2000
1.0
4 h Pulse CELLS 1500
.75
I50XI
1000
(27X) .25
500
10
20
30
40
50
60
70
80
FRACTION
FlG. 4. HPLC elution profiles of hGCTP-IR and immunoprecipitable [35S]hGCTP in pituitary cells after a 4-h pulse. Details are as in Fig. 1.
precipitable radioactivity was present in pro-hGRH. Several of the other immunoreactive peaks also contained
The present data provide evidence for the biosynthesis of hGRH in pituitaries of transgenic mice in vitro. The results have shown the incorporation of a radiolabeled amino acid (methionine) into the hGRH precursor and its processing to the mature forms of the hormone. Demonstration of this process was facilitated by the high levels (100-500 times that in rat hypothalamus) and high rate of hGRH synthesis from a transgene that is constitutively expressed in this tissue. Although the pituitary is not the normal site of GRH gene expression, it contains the necessary complement of processing enzymes to convert the prohormone to the mature hormonal peptide forms (8). The fact that the efficiency of prohormone processing is not as great as in the hypothalamus aided the present studies since it permitted greater accumulation of the prohormone and facilitated demonstration of the processing pathway. The pulse-chase study clarified several aspects of hGRH biosynthesis that had previously been assumed and/or open to speculation. First, the peak tentatively identified as the hGRH precursor (pro-hGRH) in previous studies on the basis of its molecular size and relative immunoreactivity with two separate hGRH RIAs (8) has been shown to accumulate [35S]methionine during a pulse period and to have this radiolabel diminish during a chase period. Furthermore, this peak could be demonstrated with an antibody to hGCTP as well as to hGRH. Second, the temporal sequence of accumulation of radioactivity into the two mature forms of hGRH, hGRH(l-44)-NH 2 and hGRH(l-40)-OH, indicates that the former serves as a precursor for the latter. Incorporation of radioactive label was detected in hGRH(l-44)NH2 in both cells and media by the end of the pulse period but did not appear in hGRH(l-40)-OH until the end of the first chase period. The present results provide evidence for the incorporation of radioactivity into several peaks that exhibit hGCTP immunoreactivity, one of which coelutes with pro-hGRH. The higher level of radioactivity precipitated by the hGCTP antibody as compared to the hGRH antibody may reflect different affinities of the two antibodies for pro-hGRH and/or other C terminally extended molecules. However, only a small amount of immunoprecipitable radioactivity coeluted with the synthetic hGCTP standard. These results, together with the low intracellular level of hGCTP-IR [less than 2% of hGRH(l-44)-NH 2 and hGRH(l-40)-OH on a molar basis] and the absence of detectable levels of hGCTP-IR in the incubation media (data not shown), suggest that
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 03 August 2016. at 18:49 For personal use only. No other uses without permission. . All rights reserved.
BIOSYNTHESIS OF HUMAN GRH
hGCTP, once synthesized, is rapidly degraded. The present results suggest the proposed schema of hGRH biosynthesis shown in Fig. 5. This extends the previously proposed pathways, based on the amino acid sequences of hGRH and the hGRH precursor as deduced from its cDNA (1, 2). After removal of the signal peptide, processing of pro-hGRH is initiated first by cleavage at positions 46-47 and then 45-46 by a trypsin-like endopeptidase and a carboxypeptidase, resulting in hGRH(l45)-OH and hGCTP formation. An amide group is then transferred from Gly45 to Leu44 by peptidyl glycine aamidating monooxygenase (PAM) (17). This transamidating step has recently been shown to occur using synthetic hGRH(l-45) and PAM (18, 19). Based on immunohistochemical studies using antisera specific for the carboxyl terminus of hGCTP it is likely that hGCTP also serves as a substrate for PAM, resulting in an amide transfer from Gly77 to Gin76 (5). The present study cannot resolve this possibility, since these two compounds would not be expected to be separated under the conditions employed for HPLC (8). Finally, peptidases cleave at positions 41-42 and 40-41 of hGRH(l-44)-NH 2 to generate hGRH(l-40)-OH. An obligatory role of hGRH(l-44)-NH 2 in the generation of hGRH(l-40)-OH is also suggested by the absence of both hormonal forms in the liver of transgenic hGRH mice, where there is accumulation of hGRH(l-45)-OH (8) as a consequence of the absence of PAM gene expression in this tissue (17). Some hGRH-secreting tumors also have been prcpro-GRH -30
- 1 0
Met
Arg-Arg-Tyr.
pro-GRH
1
I
40
41
44
45
46 47
77
Ala-Arg....Leu-Gly-Arg-Gln
Gly
Signal peptide cleavage
1
40
Tyr.
Ala-Arg....Leu-Gly-Arg-Gln
41
44
45
46
47
Jl
77
Gly
Endopeptidase + Carboxypeptidase
GRH(l-45)-OH
GCTP
1
40
41
44
45
Tyr.
Ala-Arg....Leu-Gly
47
77
Gin
Glv
Peptidylglycine a-amidating monooxygenase (PAM)
GRH(l-44)-NH 2 1
40
Tyr.
Ala-Arg....Leu-NH2
41
IJ
44
Endopeptidase + Carboxypeptidase
GRH(1-40)-OH 1
40
Tyr.
Ala
FIG. 5. Proposed schema for hGRH biosynthesis.
3279
shown to contain hGRH(l-37)-OH (13, 20). The present results do not clarify whether this peptide is derived by further modification of hGRH(l-40)-OH or directly from hGRH(l-44)-NH2. In the present studies there was no evidence for hGRH(l-37)-OH, which would have been expected to elute slightly beyond that of hGRH (1-40)OH. The transgenic pituitary cells and media contained several other GRH-IR peaks in addition to those that could be identified. Two major peaks, both of which incorporated considerable radioactive label, eluted just ahead of hGRH(l-44)-NH2. Neither of these peaks was recognized by the hGCTP antiserum (not shown). Their positions are consistent with those of hGRH(3-44)-NH2 and hGRH(3-40)-OH, the primary hGRH metabolites observed in plasma (21, 22). The enzyme responsible for this metabolic conversion, dipeptidylpeptidase, type IV, is not detectable in the pituitary (our unpublished observations), though this tissue does contain aminopeptidases that could possibly generate these compounds. These immunoreactive peaks have previously been noted in pituitary and, at relatively lower concentrations, in other tissues from transgenic hGRH mice (8). However, the small quantity of each of these peptides present in the pituitary precludes their absolute identification at the present time. Finally, it is of interest to note that only in the human species have two mature forms of GRH been identified. Although hGRH(l-40)-OH exhibits a longer half-life than does hGRH(l-44)-NH 2 (21), the two peptides are biologically equipotent and the physiological importance of there being two releasing hormone forms in humans, if any, is unknown. Both mouse and rat GRH contain an Arg41 (23, 24), as does hGRH, and both the mouse hypothalamus and pituitary (8, present study) contain the necessary converting enzyme(s). Since neither mouse nor rat GRH is carboxyl-terminally amidated, it is tempting to speculate that carboxyl-terminal amidation is a substrate prerequisite. However, porcine GRH, which both contains Arg41 and is carboxyl-terminally amidated (25), exists only in a 44-amino acid form. Endopeptidase cleavage at monobasic sites is believed to require specific neighboring amino acid sequences (11). The difference in carboxyl-terminal sequences of the other mammalian GRHs and hGRH (23, 26) may explain the lack of their further processing. In summary, the present pulse-chase studies have demonstrated the processing of the hGRH precursor to both mature forms of hGRH and have provided evidence that hGRH(l-40)-OH is derived from hGRH(l-44)-NH 2. It should be emphasized that the present pulse-chase studies were performed in pituitary rather than hypothalamus. Although mouse hypothalamus is capable of generating hGRH(l-40)-OH (8), the possibility cannot be
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 03 August 2016. at 18:49 For personal use only. No other uses without permission. . All rights reserved.
BIOSYNTHESIS OF HUMAN GRH
3280
excluded that the processing mechanism in this tissue varies from that in the pituitary. Nevertheless, the fact that these studies were conducted in a different species as well as tissue suggests that the processing enzymes required for human GRH biosynthesis are present in multiple tissues and functionally preserved in at least two mammalian species.
Acknowledgments We thank Jane Withrow for her excellent technical assistance.
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