The Regulation of Human P450c17 Activity: Relationship to Premature Adrenarche, Insulin Resistance and the Polycystic Ovary Syndrome

The Regulation of Human P450c17 Activity: Relationship to Premature Adrenarche, Insulin Resistance and the Polycystic Ovary Syndrome Richard J. Auchus, David H. Geller, Tim C. Lee and Walter L. Miller

The polycystic ovary syndrome (PCOS) is characterized by hyperandrogenism and insulin resistance, but the connection between these two features has been unclear. Androgen synthesis is regulated in part by the ratio of the 17α-hydroxylase and 17,20 lyase activities of P450c17. Three separate lines of evidence show that the ratio of lyase to hydroxylase activity is regulated by electron flow from P450 oxidoreductase. Lyase activity and androgen synthesis are particularly dependent on the serine phosphorylation of P450c17. Serine phosphorylation of the insulin receptor β chain causes insulin resistance, and some PCOS women have hyperphosphorylated receptors. We hypothesize that an overactive serine/threonine kinase hyperphosphorylates both the insulin receptor and P450c17 in PCOS, accounting for the characteristic insulin resistance and hyperandrogenism of this disease.

Adrenarche is the temporally programmed rise in the secretion of adrenal androgens that begins at about age eight to ten and is independent of puberty (Sklar et al. 1980). The pattern of hyperandrogenism in premature and exaggerated adrenarche resembles the overproduction of adrenal androgens seen in the polycystic ovary syndrome (PCOS) (Lachelin et al. 1979, Lucky et al. 1986), and girls with premature adrenarche are more likely to develop PCOS (Ibáñez et al. 1993, Oppenheimer et al. 1995). In both adrenarche and PCOS, adrenal secretion of C19 sex steroids increases without a concomitant increase in the secretion of adrenocorticotropic hormone (ACTH) or cortisol, implying an R.J. Auchus, D.H. Geller, T.C. Lee and W.L. Miller are at the Department of Pediatrics, Bldg MR-IV Room 209, University of California San Francisco, San Francisco, CA 94143-0978, USA.

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increase in 17,20 lyase activity. Both 17α-hydroxylase and 17,20 lyase activities are catalyzed exclusively by P450c17 (Nakajin et al. 1984): there is only one gene for P450c17 in the human genome (Picado-Leonard and Miller 1987), which encodes one species of mRNA (Chung et al. 1987), and severe mutation of this gene wholly ablates both activities (Yanase 1995). Thus, we have sought to understand the biochemical basis of the differential regulation of the 17α-hydroxylase and 17,20 lyase activities of P450c17 to illuminate the mechanisms of adrenarche and PCOS. •

P450c17

P450c17 catalyzes steroidogenic reactions by activating molecular oxygen with two electrons from NADPH that are delivered to P450 by a membranebound flavoprotein called P450 oxidoreductase (OR). Studies using purified porcine P450c17 combined with rat or

rabbit OR indicated that much higher molar ratios of OR to P450c17 were needed to elicit 17,20 lyase activity than 17α-hydroxylase activity (Onoda and Hall 1982), and that cytochrome b5 could augment the 17,20 lyase activity more effectively than the 17αhydroxylase activity (Yanagibashi and Hall 1986, Katagiri et al. 1995). Thus, it has generally been thought that b5 serves as an alternative electron donor for P450c17. When our laboratory cotransfected non-steroidogenic COS-1 cells with vectors expressing human P450c17 and OR, or P450c17 and b5, we confirmed that excess OR selectively stimulated 17,20 lyase activity but we found no effect with b5 (Lin et al. 1993). Thus, a role for b5 in human 17,20 lyase activity has been controversial, and the mechanism of any such action has been unclear. To resolve these questions, we used a series of five genetically engineered yeast strains including some in which the endogenous yeast genes for OR or b5 were disrupted, to study the effects of human b5 and/or OR on human P450c17 (Auchus et al. 1998). In yeast microsomes, both 17α-hydroxylase and 17,20 lyase activities absolutely required OR; and neither yeast OR nor human b5 in the absence of human OR could support 17,20 lyase activity. Addition of human b5, either by cotransfection of a human b5 expression vector or by addition of purified recombinant human b5 to yeast microsomes, would augment the 17,20 lyase activity of P450c17, but only if OR was present. Most interestingly, the effect of b5 added to microsomes could be reproduced with apo-b5 (i.e. cytochrome b5 lacking the heme group). Titration curves showed that the stimulatory action of b5 was maximal at a 30:1 molar ratio of b5 to P450c17, but then fell back toward control values at higher ratios. By contrast, the stimulatory action of apo-b5 was maximal at 30:1, but no inhibition was seen at higher ratios. These data suggest that both b5 and apo-b5 augment 17,20 lyase as allosteric facilitators. Electron transfer from b5 to P450c17 is not a factor, but at very high concentrations of b5 there may be reverse

© 1998, Elsevier Science Ltd, 1043-2760/98/$19.00. PII: S1043-2760(98)00016-2

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electron flow, from P450c17 to b5, accounting for the inhibition of 17,20 lyase activity with very high ratios of b5 to P450c17. Thus, the molar abundance of OR and its proper positioning with P450c17, which can be influenced by allosteric interactions, are key regulators of the ratio of the 17,20 lyase to 17α-hydroxylase activity of P450c17. •

Isolated 17,20 Lyase Deficiency

Another way of studying the differential regulation of the two activities of P450c17 is to search for mutations that selectively delete the 17,20 lyase activity and retain the 17α-hydroxylase activity. Based on our suggestion that residues 346–369 of human P450c17 constitute the substrate-binding site (Picado-Leonard and Miller 1988), Dufau’s laboratory performed sitedirected mutagenesis of this region in rat P450c17 and found that the mutation R346A retained 80% of its 17α-hydroxylase activity but only 7% of its 17,20 lyase activity, while R357A retained 35% of its 17α-hydroxylase activity but only 14% of its 17,20 lyase activity (Kitamura et al. 1991). Thus, mutation of single amino acid residues could substantially alter the enzyme’s ratio of lyase to hydroxylase activities. A human disease of isolated 17,20 lyase deficiency was first reported in 1972 (Zachmann et al. 1972), but the existence of such a disorder was called into question when one of the first cases described was studied at a molecular genetic level and found to have two wholly inactivating mutations in P450c17 (Yanase et al. 1992). We recently studied two patients from rural Brazil who had hormonal and anatomical findings consistent with a selective defect in 17,20 lyase activity (Geller et al. 1997). One patient was homozygous for the mutation R347H, the other for R358Q, which correspond exactly with the altered residues in the rat enzyme that selectively diminished lyase activity (Kitamura et al. 1991). When expressed in vitro, each human mutant retained ~65% of normal 17α-hydroxylase activity but <5% of normal 17,20 lyase activity. Substrate competition experiments showed no

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differences in 17OH-pregnenolone binding between the wild-type and mutant enzymes, indicating that the mutations are not in the active site. To determine how these mutants selectively eliminated 17,20 lyase activity, we built a molecular model of human P450c17 based on the X-ray crystal structure of three bacterial cytochrome P450 enzymes, in particular, P450-BMP (Ravichandran et al. 1993). P450-BMP is a type II P450 that interacts directly with a flavoprotein, analogously to microsomal P450s, whereas all other bacterial P450s crystallized to date are type I P450s that use an iron/ sulfur protein intermediate, similar to mitochondrial P450 enzymes. The model was built by aligning the sequences based on their predicted secondary structures, rather than based on amino acid sequence identity (Graham-Lorence and Peterson 1996). The resulting model shows that the R347 and R358 residues involved in isolated 17,20 lyase deficiency do not lie in the active site but instead lie in the redox-partner-binding site on the opposite side of the heme moiety from the substrate-binding site (Geller et al. 1997). The R347H and R358Q mutations change positive surface charges to negative ones, presumably making electrostatic interactions with OR less likely, and therefore diminishing lyase activity to a greater extent than hydroxylase activity. Thus, mutations that reduce the ability of P450c17 to interact with OR have the same effect as lowering the concentrations of OR. •

Phosphorylation of P450c17

Although an increase in the abundance of OR or b5 may increase the lyase/ hydroxylase ratio, an increase in the expression of either of these proteins should also increase 21-hydroxylation, catalyzed by microsomal P450c21, which is closely related to P450c17 (Picado-Leonard and Miller 1987). An increase in 21-hydroxylation would be seen as an increase in cortisol and aldosterone production, but this is not seen at adrenarche. Therefore, we postulated that the differential regulation of 17,20 lyase activity required a post-translational modification of

P450c17 (Zhang et al. 1995). In both human adrenocortical NCI-H295 cells, which express the endogenous P450c17 gene (Staels et al. 1993), and in COS-1 cells transfected with a P450c17 expression vector, P450c17 was rapidly phosphorylated on serine and threonine (but not tyrosine) residues in response to cAMP (but not in response to phorbol ester). Treatment of cells with cAMP or cotransfection with a vector expressing the catalytic β subunit of protein kinase A increased lyase activity slightly, but the substantial basal lyase activity made interpretation difficult. However, when human adrenal microsomes were dephosphorylated with alkaline phosphatase, their 17,20 lyase activity was reduced drastically, while the hydroxylase activity was unaffected. This dephosphorylation did not appear to alter the substrate-binding site, as the substrate-induced difference spectra were not altered. While the specific locations of the phosphorylated serine and threonine residues are not known, we hypothesize that they will be involved in facilitating the interaction of P450c17 with OR. Thus, it appears that all three lines of investigation outlined above indicate that the ratio of 17,20 lyase to 17α-hydroxylase activity of P450c17 is regulated by factors that control electron flow from OR (Figure 1). •

Insulin Resistance and PCOS

Mechanisms linking the hyperandrogenism and insulin resistance of PCOS have been debated for years, but none of the previous models has explained the available clinical data, which indicate that both of these disorders are primary events, and that many patients have a heritable, possibly autosomal dominant form of PCOS. Serine phosphorylation of the β chain of the insulin receptor has long been known to interfere with receptor tyrosine phosphorylation and thus provide an apparent mechanism for insulin resistance (Bollag et al. 1986, Stadtmauer and Rosen 1986, Takayama et al. 1988, Kahn 1994). Therefore, when we found that hyperphosphorylation of P450c17 increases androgen synthesis, TEM Vol. 9, No. 2, 1998

P450c17

Optimize conformation of P450c17–OR complex:

OR

• Serine phosphorylation • Positive charges in redox partner binding site • Abundance of OR • Presence of b5

P

FMN

+ Fe

+ + +

H

FAD Microsomal membrane

References Auchus R, Lee T, Miller W: 1998. Cytochrome b5 augments the 17,20 lyase activity of human P450c17 without direct electron transfer. J Biol Chem 273:3158–3165. Bollag G, Roth R, Beaudoin J, Mochley-Rosen D, Koshland D, Jr: 1986. Protein kinase C directly phosphorylates the insulin receptor in vitro and reduces its protein-tyrosine kinase activity. Proc Natl Acad Sci USA 83:5822–5824. Carey AH, Chan KL, Short F, et al.: 1992. Evidence for a single gene effect in polycystic ovaries and male pattern baldness. Clin Endocrinol 38:653–658.

P

b5

Chung B, Picado-Leonard J, Haniu M, et al.: 1987. Cytochrome P450c17 (steroid 17α-hydroxylase/17,20 lyase): cloning of human adrenal and testis cDNAs indicates the same gene is expressed in both tissues. Proc Natl Acad Sci USA 84:407–411.

Figure 1. Formation of the P450c17–OR complex. A recent X-ray crystal structure (Wang et al. 1997) has shown that OR is a multidomain protein in which electrons are passed from NADPH to the FAD (tricyclic structure in domain touching the membrane) in one domain to the FMN in another domain. The FMN domain flexes about a hinge region and connecting domain (H) so that the FMN moiety can approach the redox-partner-binding site (concave surface below heme) in P450c17 (Auchus et al. 1998). The 17,20 lyase reaction requires precise orientation of OR in the redox-partner-binding site of P450c17 in order for catalysis to occur. The P450c17–OR complex adopts this conformation more readily when P450c17 is serine phosphorylated, when positive charges in the redox-partner-binding site are properly distributed, and when cytochrome b5 is present at optimal molar ratios. Conversely, 17,20-lyase activity is reduced when these factors are absent or when the abundance of OR is reduced. Thus, multiple mechanisms regulate 17,20-lyase activity via a final common pathway.

Dunaif A, Xia J, Book C-B, Schenker E, Tang Z: 1995. Excessive insulin receptor serine phosphorylation in cultured fibroblasts and in skeletal muscle. J Clin Invest 96:801–810.

we suggested that a single kinase might hyperphosphorylate both the insulin receptor and P450c17, resulting in the two primary features of PCOS. At about the same time, Dunaif et al. (1995) showed that the β chain of the insulin receptor is 3.7-fold hyperphosphorylated in about half of women with PCOS diagnosed on the basis of insulin resistance and hyperandrogenism. Thus, data are accumulating to support the “serine phosphorylation model” of PCOS. Much remains to be done. If the above model is correct, the same kinase must phosphorylate both P450c17 and the β chain of the insulin receptor. However, neither the kinase that phosphorylates P450c17 nor the kinase responsible for the 3.7-fold hyperphosphorylation of the insulin receptor in PCOS patients has been identified. Presumably, these kinases are one and the same. The next question will be whether women who have

Ibáñez L, Potau N, Virdis R, et al.: 1993. Postpubertal outcome in girls diagnosed of premature pubarche during childhood: increased frequency of functional ovarian hyperandrogenism. J Clin Endocrinol Metab 76:1599–1603.

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a familial history of PCOS have a lesion in such a kinase. It is attractive to postulate an activating mutation in a kinase, as that would predict dominant inheritance, as has been suggested for PCOS (Carey et al. 1992). Finally, the precise residues that are phosphorylated on P450c17 must be identified and their mode of action explained. Presumably, they will facilitate interaction between P450c17 and OR. •

Acknowledgements

This work was supported by the National Cooperative Program for Infertility Research at UCSF (U54HD34449) and by NIH Grants R01DK37922 and R01-DK42154 (to WLM). RJA was supported by Clinical Investigator Award K08-KD02387 and DHG was supported by T32DK0716 and a fellowship from the Lawson Wilkins Pediatric Endocrine Society.

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ing protein suggest a consensus steroidbinding sequence. Mol Endocrinol 2:1145–1150. Ravichandran KG, Boddupalli SS, Hasemann CA, Peterson JA, Deisenhofer J: 1993. Crystal structure of hemoprotein domain of P450BM-3, a prototype for microsomal P450s. Science 261:731–736. Sklar CA, Kaplan SL, Grumbach MM: 1980. Evidence for dissociation between adrenarche and gonadarche: studies in patients with idiopathic precocious puberty, gonadal dysgenesis, isolated gonadotropin deficiency, and constitutionally delayed growth and adolescence. J Clin Endocrinol Metab 51:548–556. Stadtmauer L, Rosen OM: 1986. Increasing the cAMP content of IM-9 cells alters the phosphorylation state and protein kinase activity of the insulin receptor. J Biol Chem 261:3402–3407. Staels B, Hum DW, Miller WL: 1993. Regulation of steroidogenesis in NCI-H295 cells: a cellular model of the human fetal adrenal. Mol Endocrinol 7:423–433. Takayama S, White MF, Kahn CR: 1988. Phorbol ester-induced serine phosphorylation of the insulin receptor decreases its tyrosine kinase activity. J Biol Chem 263:3440–3447.

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Coming soon in TEM Xenoestrogens: The Emerging Story of Bisphenol A – N. Ben-Jonathan Thyroid Hormone Receptor Gene Knockouts – G.A. Brent and J-H. Hsu Prolactin Receptor Gene Diversity: Structure and Regulation – Z. Hu Radioiodine-induced Thyroid Cancer: Studies in the Aftermath of the Accident at Chernobyl – J. Robbins and A.B. Schneider Leptin: A Molecule Integrating Somatic Energy Stores, Energy Expenditure and Fertility – M. Rosenbaum and R.L. Leibel Calcitonin Gene-related Peptide is a Mediator of Vascular Adaptations During Hypertension in Pregnancy – C. Yallampalli and S.J. Wimalawansa

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