Proc. Nail. Acad. Sci. USA Vol. 87, pp. 2003-2007, March 1990 Biochemistry
Charge isoforms of the adrenocortical pregnenolone-binding protein: Influence of phosphorylation on isoformation and binding activity (corticotropin/steroidogenefis/zonaion) Y. C. LEE, W. J. DRISCOLL, AND C. A. STROTT* Section on Adrenal Cell Biology, Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892
Communicated by Seymour Lieberman, December 4, 1989 (received for review August 18, 1989)
Isoelectric focusing of the Mr 34,000 pregABSTRACT nenolone-binding protein (PBP) isolated from the guinea pig adrenal cortex has revealed multiple charge isoforms. Alkaline phosphatase treatment resulted in the disappearance of the pI 5.4 isoform associated with the appearance of pI 5.9 and pI 6.1 isoforms; this alteration in the charge-isoform pattern of the PBP correlated with a loss in pregnenolone-binding activity. This finding appears to be novel for intracellular steroidbinding proteins and has not been demonstrated for steroid receptors, a well-studied group of phosphoproteins. Resolution of the PBP by nondenaturing polyacrylamide gel electrophoresis produced two radioactive peaks of [3Hlpregnenolone in an equilibrium system, while only one peak was present in a nonequilibrium system, suggesting high- and low-binding affinity forms of PBP. Isoelectric focusing of highly purified PBP resolved multiple forms of Mr 34,000 protein with pI values ranging from 6.4 to 5.2. Two of the Mr 34,000 charge isoforms were isolated, and each was used to generate polyclonal antibodies; both antisera were crossreactive against all forms of Mr 34,000 PBP. Western blot analysis revealed that the PBP was present in both the fasciculata and reticularis of the adrenal cortex, though the isoform patterns were not identical for the two zones. Additionally, the pregnenolone-binding activity was =l0-fold greater in the zona reticularis. In vitro alkaline phosphatase treatment of the PBP abolished pregnenolonebinding activity and caused an alteration in the charge-isoform pattern for PBP in the zona reticularis, where pregnenolone binding is high, to resemble the pattern found for the zona fasciculata, where pregnenolone binding is low. The results indicate that phosphorylation/dephosphorylation regulates pregnenolone-binding activity and influences the pattern of the PBP isoformation. The data further suggest that the pI 5.4 isoform may be the active steroid-binding molecule. The rate-limiting step in adrenocortical steroidogenesis is the conversion of cholesterol to pregnenolone (1, 2); however, the precise mechanism by which corticotropin (ACTH) regulates the steroidogenic process is not well understood. There has been an extensive search in recent years for a noncatalytic protein that might play an important regulatory role, presumably by interacting with cholesterol. Three distinct proteins thought to function in such a manner have been described (3-5). The aforementioned proteins are not unique to the adrenal cortex, however, having been found in gonadal as well as nonsteroidogenic tissue (6-8); thus, the cholesterol-interacting proteins appear to play a more universal role in cholesterol metabolism and steroidogenesis. But what about pregnenolone, the product of the rate-limiting step? A specific adrenocortical pregnenolone-binding protein (PBP) has
been described and partially purified, albeit its precise function is not understood (9, 10). Pregnenolone, in addition to being the primary precursor of cortisol, may itself have a biological role not previously appreciated. In contrast to the cholesterol-interacting proteins, the PBP is present only in cells of the adrenal cortex that are regulated by ACTH, and is not widely distributed (unpublished data). Although the function of the PBP remains to be determined, in this paper we will present evidence describing several notable features of the PBP: that it exists in charge isoforms, that the isoform pattern and pregnenolone-binding activity are dependent upon the phosphorylation state of the protein, and that a specific charge isoform may be the active pregnenolonebinding molecule.
MATERIALS AND METHODS Preparation of Adrenocortical Cytosol. Male guinea pigs (NIH inbred strain 2) weighing 700-900 g were used. The animals were anesthetized with CO2 gas and decapitated. The adrenals were placed in 20 mM Tris-HCl, pH 7.4/1 mM EDTA (buffer A) on ice. The adrenals were homogenized in buffer A containing 0.2 mM phenylmethylsulfonyl fluoride and cytosol prepared as previously described (11). In some experiments cytosol was prepared from separated outer and inner cortical zones (12). Isolation of PBP. Cytosol was fractionated first with DEAE-cellulose and then by gel permeation chromatography using Ultrogel AcA 44 (10). The fractions containing the pregnenolone-binding activity were combined, concentrated, and, after addition of glycerol (10%o, vol/vol), stored in liquid nitrogen. The PBP was further purified by nondenaturing PAGE and HPLC. The PAGE multiphasic buffer system designated no. 2365 (13) consisted of chloride, sulfate, asparagine, and N-(j3-hydroxyethyl)morpholine; the PAGE procedure was performed as described (9). For some experiments [3H]pregnenolone (NEN) was added to the gel solution before casting to give a baseline of =1000 cpm per 1-mm slice of gel. Proteins in the gel slices containing the pregnenolone-binding activity were eluted by diffusion into buffer A and concentrated. The PAGE-purified proteins were applied to a Vydac C4 semipreparative reverse-phase column [10 mm (i.d.) x 25 cm; 5-,m particle size] equilibrated with 80o solvent A (0.1% trifluoroacetic acid in H20) and 20o solvent B (0.1% trifluoroacetic acid and 80%o acetonitrile in H20). Proteins were fractionated with a Pharmacia/LKB HPLC system using a discontinuous linear gradient of 20% B Abbreviations: ACTH, corticotropin; IEF, isoelectric focusing;
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
PBP, pregnenolone-binding protein. *To whom reprint requests should be addressed at: National Institutes of Health, Building 10, Room B1-L400, Bethesda, MD 20892. 2003
2004
Biochemistry: Lee et al.
for 15 min, 20-50% B for 25 min, 50-70o B for 120 min, and 70-100%o B for 20 min at a flow rate of 1 ml/min at 230C. Isoelectric Gel Electrophoresis and SDS/PAGE. Isoelectric focusing (IEF) in tube gels (0.3 x 16 cm) was performed, according to O'Farrell (14), without urea in the presence of 5% glycerol. The pH gradient was established in 5% acrylamide with a mixture of 3% (vol/vol) Ampholine 5-7 (LKB) and 2% (vol/vol) Bio-Lyte 3/10 (Bio-Rad). The protein samples (10 ul) were mixed with an equal volume of 15% glycerol/6% Ampholine 5-7/4% Bio-Lyte 3/10, applied to the gels, and subjected to electrophoresis at 400 V for 16 hr followed by 800 V for 1 hr at 40C. The pH gradient was determined by slicing (3-mm sections) a focusing gel that had been run without a protein sample; each section was placed in 0.5 ml of deionized water, and the pH was measured after 30 min. Isoelectric points were estimated from a plot of pH vs. distance. Second-dimension electrophoresis (SDS/ PAGE) was performed with 12% or 15% gels as described (15). Proteins separated in the second dimension were electrophoretically transferred to nitrocellulose membranes (16) in a Trans-Blot cell (Bio-Rad) and visualized with Ponceau S solution (Sigma). Standard SDS/polyacrylamide gels were silver-stained without glutaraldehyde fixation (17). Polydonal Antibody Production. Since the amount of purified antigens was in limited supply, polyclonal antibodies were generated in female rabbits by direct inoculation into popliteal lymph nodes (18). Antigen (20-40 ,ug) was emulsifed in an equal volume of Freund's complete adjuvant and injected bilaterally (1v>< In IU
6.4
34
92
pI 3
sa-
6641
opl
6.1 5.9
04
5.5
a
45
^ 31
.0
Charge isoforms of the adrenocortical pregnenolone-binding protein: Influence of phosphorylation on isoformation and binding activity (corticotropin/steroidogenefis/zonaion) Y. C. LEE, W. J. DRISCOLL, AND C. A. STROTT* Section on Adrenal Cell Biology, Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892
Communicated by Seymour Lieberman, December 4, 1989 (received for review August 18, 1989)
Isoelectric focusing of the Mr 34,000 pregABSTRACT nenolone-binding protein (PBP) isolated from the guinea pig adrenal cortex has revealed multiple charge isoforms. Alkaline phosphatase treatment resulted in the disappearance of the pI 5.4 isoform associated with the appearance of pI 5.9 and pI 6.1 isoforms; this alteration in the charge-isoform pattern of the PBP correlated with a loss in pregnenolone-binding activity. This finding appears to be novel for intracellular steroidbinding proteins and has not been demonstrated for steroid receptors, a well-studied group of phosphoproteins. Resolution of the PBP by nondenaturing polyacrylamide gel electrophoresis produced two radioactive peaks of [3Hlpregnenolone in an equilibrium system, while only one peak was present in a nonequilibrium system, suggesting high- and low-binding affinity forms of PBP. Isoelectric focusing of highly purified PBP resolved multiple forms of Mr 34,000 protein with pI values ranging from 6.4 to 5.2. Two of the Mr 34,000 charge isoforms were isolated, and each was used to generate polyclonal antibodies; both antisera were crossreactive against all forms of Mr 34,000 PBP. Western blot analysis revealed that the PBP was present in both the fasciculata and reticularis of the adrenal cortex, though the isoform patterns were not identical for the two zones. Additionally, the pregnenolone-binding activity was =l0-fold greater in the zona reticularis. In vitro alkaline phosphatase treatment of the PBP abolished pregnenolonebinding activity and caused an alteration in the charge-isoform pattern for PBP in the zona reticularis, where pregnenolone binding is high, to resemble the pattern found for the zona fasciculata, where pregnenolone binding is low. The results indicate that phosphorylation/dephosphorylation regulates pregnenolone-binding activity and influences the pattern of the PBP isoformation. The data further suggest that the pI 5.4 isoform may be the active steroid-binding molecule. The rate-limiting step in adrenocortical steroidogenesis is the conversion of cholesterol to pregnenolone (1, 2); however, the precise mechanism by which corticotropin (ACTH) regulates the steroidogenic process is not well understood. There has been an extensive search in recent years for a noncatalytic protein that might play an important regulatory role, presumably by interacting with cholesterol. Three distinct proteins thought to function in such a manner have been described (3-5). The aforementioned proteins are not unique to the adrenal cortex, however, having been found in gonadal as well as nonsteroidogenic tissue (6-8); thus, the cholesterol-interacting proteins appear to play a more universal role in cholesterol metabolism and steroidogenesis. But what about pregnenolone, the product of the rate-limiting step? A specific adrenocortical pregnenolone-binding protein (PBP) has
been described and partially purified, albeit its precise function is not understood (9, 10). Pregnenolone, in addition to being the primary precursor of cortisol, may itself have a biological role not previously appreciated. In contrast to the cholesterol-interacting proteins, the PBP is present only in cells of the adrenal cortex that are regulated by ACTH, and is not widely distributed (unpublished data). Although the function of the PBP remains to be determined, in this paper we will present evidence describing several notable features of the PBP: that it exists in charge isoforms, that the isoform pattern and pregnenolone-binding activity are dependent upon the phosphorylation state of the protein, and that a specific charge isoform may be the active pregnenolonebinding molecule.
MATERIALS AND METHODS Preparation of Adrenocortical Cytosol. Male guinea pigs (NIH inbred strain 2) weighing 700-900 g were used. The animals were anesthetized with CO2 gas and decapitated. The adrenals were placed in 20 mM Tris-HCl, pH 7.4/1 mM EDTA (buffer A) on ice. The adrenals were homogenized in buffer A containing 0.2 mM phenylmethylsulfonyl fluoride and cytosol prepared as previously described (11). In some experiments cytosol was prepared from separated outer and inner cortical zones (12). Isolation of PBP. Cytosol was fractionated first with DEAE-cellulose and then by gel permeation chromatography using Ultrogel AcA 44 (10). The fractions containing the pregnenolone-binding activity were combined, concentrated, and, after addition of glycerol (10%o, vol/vol), stored in liquid nitrogen. The PBP was further purified by nondenaturing PAGE and HPLC. The PAGE multiphasic buffer system designated no. 2365 (13) consisted of chloride, sulfate, asparagine, and N-(j3-hydroxyethyl)morpholine; the PAGE procedure was performed as described (9). For some experiments [3H]pregnenolone (NEN) was added to the gel solution before casting to give a baseline of =1000 cpm per 1-mm slice of gel. Proteins in the gel slices containing the pregnenolone-binding activity were eluted by diffusion into buffer A and concentrated. The PAGE-purified proteins were applied to a Vydac C4 semipreparative reverse-phase column [10 mm (i.d.) x 25 cm; 5-,m particle size] equilibrated with 80o solvent A (0.1% trifluoroacetic acid in H20) and 20o solvent B (0.1% trifluoroacetic acid and 80%o acetonitrile in H20). Proteins were fractionated with a Pharmacia/LKB HPLC system using a discontinuous linear gradient of 20% B Abbreviations: ACTH, corticotropin; IEF, isoelectric focusing;
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
PBP, pregnenolone-binding protein. *To whom reprint requests should be addressed at: National Institutes of Health, Building 10, Room B1-L400, Bethesda, MD 20892. 2003
2004
Biochemistry: Lee et al.
for 15 min, 20-50% B for 25 min, 50-70o B for 120 min, and 70-100%o B for 20 min at a flow rate of 1 ml/min at 230C. Isoelectric Gel Electrophoresis and SDS/PAGE. Isoelectric focusing (IEF) in tube gels (0.3 x 16 cm) was performed, according to O'Farrell (14), without urea in the presence of 5% glycerol. The pH gradient was established in 5% acrylamide with a mixture of 3% (vol/vol) Ampholine 5-7 (LKB) and 2% (vol/vol) Bio-Lyte 3/10 (Bio-Rad). The protein samples (10 ul) were mixed with an equal volume of 15% glycerol/6% Ampholine 5-7/4% Bio-Lyte 3/10, applied to the gels, and subjected to electrophoresis at 400 V for 16 hr followed by 800 V for 1 hr at 40C. The pH gradient was determined by slicing (3-mm sections) a focusing gel that had been run without a protein sample; each section was placed in 0.5 ml of deionized water, and the pH was measured after 30 min. Isoelectric points were estimated from a plot of pH vs. distance. Second-dimension electrophoresis (SDS/ PAGE) was performed with 12% or 15% gels as described (15). Proteins separated in the second dimension were electrophoretically transferred to nitrocellulose membranes (16) in a Trans-Blot cell (Bio-Rad) and visualized with Ponceau S solution (Sigma). Standard SDS/polyacrylamide gels were silver-stained without glutaraldehyde fixation (17). Polydonal Antibody Production. Since the amount of purified antigens was in limited supply, polyclonal antibodies were generated in female rabbits by direct inoculation into popliteal lymph nodes (18). Antigen (20-40 ,ug) was emulsifed in an equal volume of Freund's complete adjuvant and injected bilaterally (1v>< In IU
6.4
34
92
pI 3
sa-
6641
opl
6.1 5.9
04
5.5
a
45
^ 31
.0
