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A serum prolactin-binding protein: implications for growth hormone
Research Update
TRENDS in Endocrinology & Metabolism Vol.12 No.10 December 2001
427
Research News
A serum prolactin-binding protein: implications for growth hormone Priscilla S. Dannies Many membrane receptors have truncated soluble forms that circulate in the blood, and a protein in serum with characteristics of the extracellular domain of the human prolactin receptor has recently been identified. Because the extracellular domain of the prolactin receptor binds human growth hormone, does the prolactin-binding protein in serum affect the amount of bound circulating growth hormone? A more general question is whether this serum protein has a minor or a major effect on the biological activities of growth hormone and prolactin.
Soluble forms have been identified for an increasing number of membrane receptors, including receptors for interleukins 1, 2 and 6, ciliary neutrophic factor, nerve growth factor, tumor necrosis factor-α, intercellular adhesion molecule (ICAM), transferrin, urokinase, growth hormone and leptin1. These forms are generated in two ways (Fig. 1): (1) through alternate splicing of mRNA for the receptor, eliminating the sequences that retain the receptor in the membrane; and (2) through proteolytic cleavage of the extracellular domain of the receptor after its synthesis. The soluble form of the growth hormone receptor, called growth hormone-binding protein, is generated by alternate mRNA splicing in rats, and by proteolytic cleavage in humans2. A soluble form of the human prolactin receptor has recently been identified in serum3, based on (1) the ability to precipitate a protein from serum the size of the extracellular domain of the human prolactin receptor prepared in bacteria, with the use of an anti-human prolactin receptor antiserum, (2) the identification by matrix-assisted laser desorption/ionization mass spectroscopy of two tryptic peptides of the human prolactin receptor obtained from the immunoprecipitate, (3) the demonstration that the protein binds human prolactin and human growth hormone. In addition, the affinity of the http://tem.trends.com
serum prolactin-binding protein for human prolactin is 13 nM (Ref. 3), in reasonable agreement with the affinity of the recombinant extracellular domain of the human prolactin receptor for human prolactin, 2 nM (Ref. 4). The sequence and origin of this serum prolactin-binding protein has not yet been characterized in detail; soluble receptors that originate by proteolytic cleavage have the same sequence as the membrane-bound receptor, but are truncated, whereas soluble receptors that derive from alternate splicing might contain some sequences that differ from the membranebound form. It is clear, however, that this serum prolactin-binding protein is similar to the prolactin receptor. The recombinant extracellular domain of the human prolactin receptor binds prolactin and growth hormone, but the binding of growth hormone is complicated by a strong Zn2+ dependency; the affinity is about 8000-fold higher in the presence of 50 µM Zn2+ than in its absence4, and His188 in the extracellular domain of the prolactin receptor is required for growth hormone binding4. If the serum human
prolactin-binding protein is a truncated form of the prolactin receptor, its ability to bind human growth hormone should also depend on Zn2+. Kline and Clevenger washed the complex with EDTA, which binds divalent cations, before identifying bound growth hormone3, which suggests that Zn2+ is not necessary. More extensive treatment with EDTA, however, might be required to remove Zn2+ and to cause the prolactin-binding protein–growth hormone complex to dissociate. Kline and Clevenger found that 50% of the growth hormone present in the serum of a single donor was bound by the serum prolactin-binding protein3. That this amount would be bound by the prolactinbinding protein might not have been expected, based on previous investigations of the state in which growth hormone exists in the circulation: about 45% is bound to growth hormone-binding protein (the soluble receptor of growth hormone), a few percent to lower affinity sites and the rest is free2. One possibility is that previous investigations that measured the amount of free growth hormone underestimated the amount of growth
(a)
(b) P
P
Nucleus
Endoplasmic reticulum
Plasma membrane TRENDS in Endocrinology & Metabolism
Fig. 1. Two mechanisms for forming soluble receptors. (a) Alternate RNA splicing. Splicing of messenger RNA in the nucleus at alternate splice sites produces some mRNAs that lack coding sequences for the amino acid sequences responsible for retaining the receptor in the membrane. The receptors synthesized from the alternatively spliced mRNA will be soluble in the endoplasmic reticulum and released from the cell when delivered to the plasma membrane. Receptors coded for by the alternatively spliced message will have the same amino acid sequences as full length receptors in the N-terminal portion of the extracellular domain, but there could be differences in sequences at the C-terminal end, coded by the alternate exon. (b) Proteolytic cleavage. Receptors are all transmembrane proteins after synthesis, but subsequently the extracellular domain is cleaved by a protease (P), most likely to be located on the plasma membrane, which allows most of the extracellular domain to become soluble.
1043-2760/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S1043-2760(01)00497-0
428
Research Update
hormone bound to the serum prolactinbinding protein because of the absence of Zn2+ in those assays, and that most growth hormone in serum is really bound to proteins, and not free. Another possibility is that the donor does not have the average distribution of bound growth hormone; variation among individuals has been observed2. Soluble receptors affect the biological activity of the ligands that they bind in at least two ways. One is that they prolong the circulation time of many growth factors and hormones, and therefore provide a more stable pool of hormone which extends the biological activity. The second effect is that they reduce the effective concentrations at the membrane receptor by competing with that receptor for binding to the hormone, which may
TRENDS in Endocrinology & Metabolism Vol.12 No.10 December 2001
reduce the biological activity1,2. These two competing effects make it difficult to predict what the overall physiological effect is in different situations. The presence of the human prolactin-binding protein will complicate efforts to understand what occurs with growth hormone, for in the presence of Zn2+, the extracellular domain of the human prolactin receptor binds human growth hormone with up to 100-fold higher affinity than human prolactin, and 10-fold higher than human growth hormone binds to the extracellular domain of its receptor4. The serum prolactin-binding protein therefore can be a potent growth hormone-binding protein. The biological effects of the serum prolactin-binding protein, whatever they turn out to be, might be as marked for the biological
activities of growth hormone as they are for those of prolactin. References 1 Rose-John, S. and Heinrich, P.C. (1994) Soluble receptors for cytokines and growth factors: generation and biological function. Biochem. J. 300, 281–290 2 Baumann, G. (2001) Growth hormone binding protein 2001. J. Pediatr. Endocrinol. Metab. 14, 355–375 3 Kline, J.B. and Clevenger, C.V. (2001) Identification and characterization of the prolactin-binding protein in human serum and milk. J. Biol. Chem. 276, 24760–24766 4 Cunningham, B.C. et al. (1990) Zinc mediation of the binding of human growth hormone to the human prolactin receptor. Science 250, 1709–1712
Priscilla S. Dannies Dept of Pharmacology, Yale School of Medicine, New Haven, CT 06520-8066, USA. e-mail: [email protected]
Leptin and puberty Henryk F. Urbanski Leptin is thought to relay metabolic information to the hypothalamic–pituitary– gonadal axis and to participate in the neuroendocrine control of puberty. To help elucidate the underlying mechanism, Cheung et al. recently performed a diverse series of experiments, the results of which undermine the prevailing hypothesis that leptin acts as a metabolic trigger for the initiation of puberty. Instead, their results suggest that leptin is one of many permissive metabolic factors that allow pubertal development to proceed.
The discovery of the adipose tissue hormone, leptin, seven years ago represents a milestone in our understanding of how body mass and energy balance affect reproductive function1. Before this discovery, it was already well established that sexual maturation does not normally occur until the body reaches a certain level of somatic development, but the underlying mechanism was poorly understood and the identity of the key neuroendocrine mediators was unclear. This changed dramatically once it was shown that leptin, a protein product of the obese gene (Lep), relays information about the state of the adipose tissue mass to the feeding behavior centers of the brain. More importantly, clinical data began to emerge showing that http://tem.trends.com
circulating leptin concentrations in boys surge just before the onset of puberty, and this raised the exciting possibility that leptin might also be physiologically involved in the maturation of the reproductive axis. Nevertheless, whether leptin acts as the primary trigger for the initiation of puberty or whether it simply acts in a permissive way to allow pubertal maturation to proceed is still highly debatable2–5. To help resolve this issue, Cheung et al.6 recently performed a series of well-orchestrated experiments, using rodents, in which they examined the following specific questions: (1) Does the developmental rise in circulating leptin concentrations actually precede the onset of puberty? (2) Does the expression of leptin receptor mRNA in the hypothalamus increase during puberty? (3) Can treatment with leptin advance puberty onset? Plasma leptin concentrations
If leptin plays a key role in triggering the onset of puberty, a significant increase in plasma leptin concentrations should take place during early pubertal development. In most (but not all) of the correlative clinical studies that have been reported, a prepubertal rise in circulating leptin concentrations was indeed observed. To examine this issue in rodents, Cheung et al. collected terminal blood samples
from developing postnatal male and female rats at five-day intervals and, as expected, the animals showed a steady increase in body mass during postnatal development. However, serum leptin concentrations did not show a significant increase until after the animals had become adults. Although it is possible that an earlier (prepubertal) increase might have been detected by collection of blood samples later in the day (leptin secretion is known to be higher at night than during the day7 and the pubertal increase in serum leptin concentrations might initially be confined to the night, as is the case for luteinizing hormone), a more conservative interpretation of the data is that leptin does not act as a principal trigger for puberty onset in rats. Leptin receptor mRNA
If the onset of puberty is preceded by an increase in the sensitivity of the hypothalamic–pituitary–gonadal axis to leptin, then leptin could still act as a pubertal trigger in spite of the absence of an obvious prepubertal rise in plasma leptin concentrations. To address this possibility, Cheung et al. performed a second experiment in which in situ hybridization histochemistry was used to measure expression of mRNA encoding the leptin receptor (Lepr) in the
1043-2760/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S1043-2760(01)00505-7
TRENDS in Endocrinology & Metabolism Vol.12 No.10 December 2001
427
Research News
A serum prolactin-binding protein: implications for growth hormone Priscilla S. Dannies Many membrane receptors have truncated soluble forms that circulate in the blood, and a protein in serum with characteristics of the extracellular domain of the human prolactin receptor has recently been identified. Because the extracellular domain of the prolactin receptor binds human growth hormone, does the prolactin-binding protein in serum affect the amount of bound circulating growth hormone? A more general question is whether this serum protein has a minor or a major effect on the biological activities of growth hormone and prolactin.
Soluble forms have been identified for an increasing number of membrane receptors, including receptors for interleukins 1, 2 and 6, ciliary neutrophic factor, nerve growth factor, tumor necrosis factor-α, intercellular adhesion molecule (ICAM), transferrin, urokinase, growth hormone and leptin1. These forms are generated in two ways (Fig. 1): (1) through alternate splicing of mRNA for the receptor, eliminating the sequences that retain the receptor in the membrane; and (2) through proteolytic cleavage of the extracellular domain of the receptor after its synthesis. The soluble form of the growth hormone receptor, called growth hormone-binding protein, is generated by alternate mRNA splicing in rats, and by proteolytic cleavage in humans2. A soluble form of the human prolactin receptor has recently been identified in serum3, based on (1) the ability to precipitate a protein from serum the size of the extracellular domain of the human prolactin receptor prepared in bacteria, with the use of an anti-human prolactin receptor antiserum, (2) the identification by matrix-assisted laser desorption/ionization mass spectroscopy of two tryptic peptides of the human prolactin receptor obtained from the immunoprecipitate, (3) the demonstration that the protein binds human prolactin and human growth hormone. In addition, the affinity of the http://tem.trends.com
serum prolactin-binding protein for human prolactin is 13 nM (Ref. 3), in reasonable agreement with the affinity of the recombinant extracellular domain of the human prolactin receptor for human prolactin, 2 nM (Ref. 4). The sequence and origin of this serum prolactin-binding protein has not yet been characterized in detail; soluble receptors that originate by proteolytic cleavage have the same sequence as the membrane-bound receptor, but are truncated, whereas soluble receptors that derive from alternate splicing might contain some sequences that differ from the membranebound form. It is clear, however, that this serum prolactin-binding protein is similar to the prolactin receptor. The recombinant extracellular domain of the human prolactin receptor binds prolactin and growth hormone, but the binding of growth hormone is complicated by a strong Zn2+ dependency; the affinity is about 8000-fold higher in the presence of 50 µM Zn2+ than in its absence4, and His188 in the extracellular domain of the prolactin receptor is required for growth hormone binding4. If the serum human
prolactin-binding protein is a truncated form of the prolactin receptor, its ability to bind human growth hormone should also depend on Zn2+. Kline and Clevenger washed the complex with EDTA, which binds divalent cations, before identifying bound growth hormone3, which suggests that Zn2+ is not necessary. More extensive treatment with EDTA, however, might be required to remove Zn2+ and to cause the prolactin-binding protein–growth hormone complex to dissociate. Kline and Clevenger found that 50% of the growth hormone present in the serum of a single donor was bound by the serum prolactin-binding protein3. That this amount would be bound by the prolactinbinding protein might not have been expected, based on previous investigations of the state in which growth hormone exists in the circulation: about 45% is bound to growth hormone-binding protein (the soluble receptor of growth hormone), a few percent to lower affinity sites and the rest is free2. One possibility is that previous investigations that measured the amount of free growth hormone underestimated the amount of growth
(a)
(b) P
P
Nucleus
Endoplasmic reticulum
Plasma membrane TRENDS in Endocrinology & Metabolism
Fig. 1. Two mechanisms for forming soluble receptors. (a) Alternate RNA splicing. Splicing of messenger RNA in the nucleus at alternate splice sites produces some mRNAs that lack coding sequences for the amino acid sequences responsible for retaining the receptor in the membrane. The receptors synthesized from the alternatively spliced mRNA will be soluble in the endoplasmic reticulum and released from the cell when delivered to the plasma membrane. Receptors coded for by the alternatively spliced message will have the same amino acid sequences as full length receptors in the N-terminal portion of the extracellular domain, but there could be differences in sequences at the C-terminal end, coded by the alternate exon. (b) Proteolytic cleavage. Receptors are all transmembrane proteins after synthesis, but subsequently the extracellular domain is cleaved by a protease (P), most likely to be located on the plasma membrane, which allows most of the extracellular domain to become soluble.
1043-2760/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S1043-2760(01)00497-0
428
Research Update
hormone bound to the serum prolactinbinding protein because of the absence of Zn2+ in those assays, and that most growth hormone in serum is really bound to proteins, and not free. Another possibility is that the donor does not have the average distribution of bound growth hormone; variation among individuals has been observed2. Soluble receptors affect the biological activity of the ligands that they bind in at least two ways. One is that they prolong the circulation time of many growth factors and hormones, and therefore provide a more stable pool of hormone which extends the biological activity. The second effect is that they reduce the effective concentrations at the membrane receptor by competing with that receptor for binding to the hormone, which may
TRENDS in Endocrinology & Metabolism Vol.12 No.10 December 2001
reduce the biological activity1,2. These two competing effects make it difficult to predict what the overall physiological effect is in different situations. The presence of the human prolactin-binding protein will complicate efforts to understand what occurs with growth hormone, for in the presence of Zn2+, the extracellular domain of the human prolactin receptor binds human growth hormone with up to 100-fold higher affinity than human prolactin, and 10-fold higher than human growth hormone binds to the extracellular domain of its receptor4. The serum prolactin-binding protein therefore can be a potent growth hormone-binding protein. The biological effects of the serum prolactin-binding protein, whatever they turn out to be, might be as marked for the biological
activities of growth hormone as they are for those of prolactin. References 1 Rose-John, S. and Heinrich, P.C. (1994) Soluble receptors for cytokines and growth factors: generation and biological function. Biochem. J. 300, 281–290 2 Baumann, G. (2001) Growth hormone binding protein 2001. J. Pediatr. Endocrinol. Metab. 14, 355–375 3 Kline, J.B. and Clevenger, C.V. (2001) Identification and characterization of the prolactin-binding protein in human serum and milk. J. Biol. Chem. 276, 24760–24766 4 Cunningham, B.C. et al. (1990) Zinc mediation of the binding of human growth hormone to the human prolactin receptor. Science 250, 1709–1712
Priscilla S. Dannies Dept of Pharmacology, Yale School of Medicine, New Haven, CT 06520-8066, USA. e-mail: [email protected]
Leptin and puberty Henryk F. Urbanski Leptin is thought to relay metabolic information to the hypothalamic–pituitary– gonadal axis and to participate in the neuroendocrine control of puberty. To help elucidate the underlying mechanism, Cheung et al. recently performed a diverse series of experiments, the results of which undermine the prevailing hypothesis that leptin acts as a metabolic trigger for the initiation of puberty. Instead, their results suggest that leptin is one of many permissive metabolic factors that allow pubertal development to proceed.
The discovery of the adipose tissue hormone, leptin, seven years ago represents a milestone in our understanding of how body mass and energy balance affect reproductive function1. Before this discovery, it was already well established that sexual maturation does not normally occur until the body reaches a certain level of somatic development, but the underlying mechanism was poorly understood and the identity of the key neuroendocrine mediators was unclear. This changed dramatically once it was shown that leptin, a protein product of the obese gene (Lep), relays information about the state of the adipose tissue mass to the feeding behavior centers of the brain. More importantly, clinical data began to emerge showing that http://tem.trends.com
circulating leptin concentrations in boys surge just before the onset of puberty, and this raised the exciting possibility that leptin might also be physiologically involved in the maturation of the reproductive axis. Nevertheless, whether leptin acts as the primary trigger for the initiation of puberty or whether it simply acts in a permissive way to allow pubertal maturation to proceed is still highly debatable2–5. To help resolve this issue, Cheung et al.6 recently performed a series of well-orchestrated experiments, using rodents, in which they examined the following specific questions: (1) Does the developmental rise in circulating leptin concentrations actually precede the onset of puberty? (2) Does the expression of leptin receptor mRNA in the hypothalamus increase during puberty? (3) Can treatment with leptin advance puberty onset? Plasma leptin concentrations
If leptin plays a key role in triggering the onset of puberty, a significant increase in plasma leptin concentrations should take place during early pubertal development. In most (but not all) of the correlative clinical studies that have been reported, a prepubertal rise in circulating leptin concentrations was indeed observed. To examine this issue in rodents, Cheung et al. collected terminal blood samples
from developing postnatal male and female rats at five-day intervals and, as expected, the animals showed a steady increase in body mass during postnatal development. However, serum leptin concentrations did not show a significant increase until after the animals had become adults. Although it is possible that an earlier (prepubertal) increase might have been detected by collection of blood samples later in the day (leptin secretion is known to be higher at night than during the day7 and the pubertal increase in serum leptin concentrations might initially be confined to the night, as is the case for luteinizing hormone), a more conservative interpretation of the data is that leptin does not act as a principal trigger for puberty onset in rats. Leptin receptor mRNA
If the onset of puberty is preceded by an increase in the sensitivity of the hypothalamic–pituitary–gonadal axis to leptin, then leptin could still act as a pubertal trigger in spite of the absence of an obvious prepubertal rise in plasma leptin concentrations. To address this possibility, Cheung et al. performed a second experiment in which in situ hybridization histochemistry was used to measure expression of mRNA encoding the leptin receptor (Lepr) in the
1043-2760/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S1043-2760(01)00505-7