0013-7227/91/1295-2512$03.00/0 Endocrinology Copyright © 1991 by The Endocrine Society
Vol. 129, No. 5 Printed in U.S.A.
Brain Peptide YY Receptors: Highly Conserved Characteristics throughout Vertebrate Evolution* MINORU OKITA, AKIO INUI, YOSHIAKI HIROSUE, MASAKI MIURA, MASAHARU NAKAJIMA, AND MASATO KASUGA Second Department of Internal Medicine, Kobe University School of Medicine, Kobe, Japan
ABSTRACT. We have shown previously that peptide YY (PYY) receptors are uniquely distributed in various mammalian brains and also have identified the receptor from porcine hippocampal membranes as a protein of 50,000 mol wt. To extend these observations, both the characteristics of PYY-receptor interaction and the structure of the receptor have been examined and compared with those of its sister peptide, neuropeptide Y (NPY), in the brains of various vertebrates including mammals (human, dog, guinea pig, rat, and mouse), birds (chicken), reptiles (snapping turtle), amphibians (bullfrog), and fish (yellowtail fish). The affinities and relative potencies of PYY as well as NPY receptors for pancreatic polypeptide (PP) family peptides were about the same in all species examined except for chickens. PYY and NPY bound to both the PYY and NPY receptors with high affinities, but porcine and avian PPs did not. In chicken brain, however, PYY, NPY, porcine PP, and avian PP all bound to the receptors with high affinity. Analysis of the equilibrium binding data for PYY receptors produced curvilinear Scatchard plots in all of the species, suggesting the existence of high and
low affinity binding sites. Affinity cross-linking using disuccinimidyl suberate followed by electrophoretic analysis of ligandreceptor complexes characterized the molecular size of PYY and NPY receptors. [125I]PYY was cross-linked to a protein of 50,000 mol wt without sulfhydryl-bonded subunits on mammalian hippocampal membranes. A receptor protein with the same mol wt was identified in other brain areas, including hypothalamus and pituitary. PYY receptors in other vertebrate brains were similar in size to those of mammalian species except in chicken brain, where a receptor protein of 67,000 mol wt was observed. In addition, we also have demonstrated that the NPY receptor is a monomeric 50,000 and 55,000 mol wt protein in mammalian and fish brains, respectively. These findings indicate that brain PYY and NPY receptors in most vertebrate species from fish to man are pharmacologically and structurally similar and have been well conserved over a period of evolution of 400 million yr. The divergence of the receptors observed in chicken brain may reflect some change in their function. {Endocrinology 129: 2512-2520, 1991)
P
EPTIDE YY (PYY), named for its C-terminal and N-terminal tyrosine residues, is a 36-amino acid peptide originally isolated from porcine duodenum (1). This peptide is a member of a family of structurally related peptides which also includes pancreatic polypeptide (PP), a pancreatic hormone (2, 3), and neuropeptide Y (NPY), a neuropeptide important in both the central and peripheral nervous systems (4). Among the porcine peptides, identities of PYY sequences with those of PP and NPY are 50 and 69%, respectively. The primary structures of porcine, rat (5), and human (6) PYY are now known. The only differences are at positions 3 and 18, where the isoleucine and asparagine residues of the human peptide are replaced by the alanine and serine residues in the porcine and rat peptides. Although PYY is found in endocrine cells of the gut and is known to Received June 4,1991. Address all correspondence and requests for reprints to: Dr. Akio Inui, Second Department of Internal Medicine, Kobe University School of Medicine, Kusunoki-cho 7-5-1, Chuo-ku, Kobe 650, Japan. * This work was supported in part by Ministry of Education, Science, and Culture of Japan Grant-in-Aid for Special Project Research 01570642.
have diverse effects on gastrointestinal function (7, 8), recent studies have revealed small quantities of PYYlike material in mammalian (9,10) and lower vertebrate (11) brains. PYY-like immunoreactivity has a topographically unique distribution and does not exhibit any overlap with other known peptide distribution, including that of NPY, suggesting a possible anatomical basis for the effects of PYY on brain function. In fact, central administration of PYY influences pituitary hormone release, thermoregulation, feeding behavior, and learning (1214). A necessary prerequisite to the biological action of peptides is an interaction with specific receptors. PYY and NPY receptors have been characterized in central and peripheral tissues and in a variety of neuronal cell lines (15-24). Furthermore, recent studies have revealed two distinct subtypes of the receptors for these peptides (21, 23, 25). Using porcine hippocampal membranes, we have demonstrated previously that PYY and NPY but not PP could bind to both the PYY and NPY receptors with great specificity (17,19). Affinity cross-linking studies also showed both brain receptors to be proteins of
2512
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 27 November 2015. at 16:00 For personal use only. No other uses without permission. . All rights reserved.
EVOLUTION OF BRAIN PYY RECEPTORS 50,000 mol wt with no subunit structures (19). However, it remains unclear whether these receptors are conserved in man or in more primitive vertebrates. No systematic attempt has yet been made to investigate the evolution of these receptors. The objective of the present study is to extend our observations in the brains of various vertebrate species from fish to man in order to understand the evolution of the receptors for PP family peptides.
Materials and Methods Reagents Synthetic porcine PYY, porcine NPY, and avian PP (APP) were purchased from Peninsula Laboratories (Belmont, CA); highly purified porcine PP (PPP) from Novo (Bagsvaerdt, Denmark); and carrier-free Na[126I] and [125I]-Bolton-HunterNPY from Amersham (Tokyo, Japan). Chloramine-T, bacitracin, phenylmethylsulfonyl fluoride (PMSF), BSA fraction V, and 2-mercaptoethanol (2ME) were obtained from Wako Pure Chemical Industries (Osaka, Japan); Sephadex G-50 superfine from Pharmacia Fine Chemicals (Tokyo, Japan); disuccinimidyl suberate (DSS) from Pierce Chemicals (Rockford, IL); mol wt standards for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) from Bio-Rad (Tokyo, Japan); and Kodak X-Omat AR films from Eastman Kodak (Rochester, NY). All other chemicals were of analytical grade. Preparation of brain membranes Human brain was obtained at postmortem from a male patient with no clinical manifestation of neurological diseases. Hartley guinea pigs, Sprague-Dawley rats, and C57BL/6 mice obtained from Japan SLC Co. Ltd. (Shizuoka, Japan) were decapitated, and their brains were rapidly removed. Brains were frozen, and hippocampi were dissected. Porcine brains were obtained from a local slaughterhouse, frozen, and dissected into hippocampi, amygdalas, caudate nuclei, and pituitary glands. Adult mongrel dogs were anesthetized with sodium thiamylal and perfused with 0.9% saline containing aprotinin. Brains were removed immediately after killing, frozen rapidly, and dissected into hippocampi, hypothalami, and pituitary glands. Chicken cerebellum and whole brains of snapping turtles, bullfrogs, and yellowtail fish were obtained soon after decapitation and frozen until used. Whole brains were used to obtain enough tissue for these species. Brain membranes were prepared as previously described (17). Briefly, brain tissues were homogenized in 5 vol ice-cold 234 mM sucrose with a motor-driven Teflon-glass homogenizer (Yamamoto Factory, Kyoto, Japan). Homogenates were centrifuged at 700 X g for 10 min to remove cellular and nuclear debris, and the supernatants were then centrifuged at 100,000 X g for 20 min at 4 C. Pellets from this spin were washed with 25 mM Tris-HCl buffer (pH 7.4) containing 1 mM PMSF and 1 mg/ml bacitracin, centrifuged again, and resuspended by homogenization in the same buffer. Membrane protein concentration was determined by the method of Bradford (26), using bovine immunoglobulin G as a standard.
2513
Receptor binding studies Synthetic porcine PYY was radioiodinated to a specific activity of 60-80 fiC'i/ng by a modification of the chloramine-T method and purified by chromatography on Sephadex G-50 superfine as described elsewhere (27). Standard incubation buffer consisted of 25 mM Tris-HCl (pH 7.4), 10 mM MgCl2) 1 mM PMSF, 1 mg/ml bacitracin, and 5 mg/ml BSA. Brain membranes at 200 /xg protein/ml were incubated in a final vol of 0.5 ml incubation buffer with 40 pM [125I]PYY for 180 min at 25 C. Binding of [126I]-Bolton-Hunter-NPY (SA, 500 /xCi/ jig) was performed similarly, except that brain membranes were incubated with 5 pM radioligand for 90 min at 25 C. Bound and free peptides were separated by centrifugation at 10,000 X g for 3 min at 4 C. Specific binding was calculated as the difference in radioactivity bound in the presence and absence of 1 /iM unlabeled PYY or NPY. Degradation of radiolabeled peptides was assessed by precipitation with trichloroacetic acid and was routinely less than 5% at the termination of incubation. Displacement data were analyzed by computerized Scatchard analysis (28). Affinity cross-linking studies Brain membranes at 400 ng protein/ml were incubated in a final vol of 1 ml 50 mM HEPES-KOH buffer (pH 7.6) containing 1 mM MgCl2, 1 mM PMSF, and 1 mg/ml bacitracin, with about 500 pM [125I]PYY or 60 pM[125I]-Bolton-Hunter-NPY for 120 min at 25 C. The receptor-bound radioligand was separated by centrifugation at 10,000 X g for 3 min, washed twice, and resuspended in 0.9 ml of the same buffer. DSS, freshly dissolved in dimethyl sulfoxide, was added to give a final concentration of 1 mM, and the cross-linking was allowed to proceed for 30 min on ice. The reaction was then quenched with 50 mM TrisHCl buffer (pH 7.6) containing 1 mM MgCl2,1 mM PMSF, and 1 mg/ml bacitracin. The cross-linked receptor-ligand complex was pelleted by centrifugation and solubilized with 50 ^1 10% SDS and 20 fA 20% glycerol in either the presence or absence of 2ME. SDS-PAGE was performed according to Laemmli (29), using 2-mm thick slab gels containing 10% acrylamide. The gels were fixed in 7% acetic acid. After visualizing the proteins with Coomassie brilliant blue, the gels were destained and dried. Autoradiograms were made from dried gels by exposure to Kodak X-Omat AR films at -80 C.
Results Pharmacological characterization of PYY and NPY binding to vertebrate brain membrane receptors The most important characterizing feature of PYY receptors in porcine hippocampus is their ability to distinguish PYY and NPY from other related or unrelated peptides (17, 19). In all species examined, [125I]PYY binding to brain membranes was inhibited by PYY and NPY in a concentration-dependent manner. PPs (APP and PPP), however, were essentially unable to inhibit [125I]PYY binding in any vertebrate species except birds. (Fig. 1) Scatchard analysis of the equilibrium binding data in
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 27 November 2015. at 16:00 For personal use only. No other uses without permission. . All rights reserved.
2514
EVOLUTION OF BRAIN PYY RECEPTORS
Endo• 1991 Voll29«No5
Chicken
Human
10 9 8 7 Peptide. - L o g (M)
10 9 8 7 Peptide. -Log (M)
Turtle
Guinea
APP FIG. 1. Inhibition of [126I]PYY binding
to brain membrane preparations by unlabeled PYY and structurally related peptides. Membranes were obtained from human, guinea pig, rat, and mouse hippocampi, chicken cerebellum, and snapping turtle, bullfrog, and yellowtail fish brains as described in Materials and Methods and were incubated with 40 pM [126I]PYY for 180 min at 25 C. Varying concentrations of PYY (•), NPY (O), APP (A), and PPP (A) were also present, as shown. Nonspecific binding was subtracted from total binding, and specific binding was expressed as a percentage of maximal specific binding. Each point is the mean ± SEM of triplicate determinations from a representative experiment.
11
7 10 9 8 Peptide. -Log (M)
12
11
10 9 8 7 Peptide. -Log (M) Frog APP
12
11
10 9 8 7 Peptide. -Log (M)
12
11
10
T
Peptide. -Log (M)
Mouse
Fish
Peptide. -Log (M) 10
Peptide. -Log (M) 11
Fig. 1 appears in Fig. 2. Scatchard plots for PYY binding 4.8 x 10" M for turtle, 1.8 X 10" M for frog, and 1.9 X to vertebrate brain membranes were, in every case, cur10~n M for fish. Total binding capacities calculated for vilinear, suggesting the existence of high and low affinity the high affinity component in the respective species components of [125I]PYY binding. The high affinity comwere 0.064, 0.022, 0.14, 0.018, 0.24, 0.17, 0.008, and 0.009 pmol/mg protein. Inhibition constants (K;) (30) for PYY ponent of PYY binding showed a similar affinity from fish to man; dissociation constants were 4.8 X 10~n M and NPY were 1.4 X 10"11 and 2.6 X 10"11 M for human; n 10 for human, 8.4 x 10~ M for guinea pig, 2.8 X 10~ M 6.1 x 10"11 and 3.3 x 10"10 M for guinea pig; 1.8 X 10"10 for rat, 4.1 x 10~n M for mouse, 2.2 x 10~10 M for chicken, and 1.8 x 10"9 M for rat; 9.6 x 10" n and 1.9 X 10~10 M
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 27 November 2015. at 16:00 For personal use only. No other uses without permission. . All rights reserved.
EVOLUTION OF BRAIN PYY RECEPTORS U. 1 J
2515 0.30
Human
Chicken
i
IT \
0.10
c 3
o
0.05
CD
1 2 6 PYY Bound, pmol/mg protein
1 2 3 PYY Bound, pmol/mg protein 0.06 o i?
0.15
Guinea Pig
Turtle
0.04
C 3 co 0.02
FIG. 2. Scatchard analysis of [125I]PYY binding to mammalian and nonmammalian brain membranes. Displacement data from Fig. 1 were subjected to the analysis, and the bound to free ratio of PYY was plotted against bound PYY per milligram of protein.
2
10
20
30
2 10 20 30 PYY Bound, pmol/mg protein
PYY Bound, p m o l / m g protein 0.-06
0.06
Rat
Frog
£ 0. 04 3
& 0 02
1
2 " 8
PYY Bound, p m o l / m g protein
jnd/Free
0 .06
o CD
0
Mouse
1 4
1 10 20 PYY Bound, pmol/mg protein
Fish
0
^>—^ 1
«
2 " 10
20
30
PYY Bound. pmol/mg protein
for mouse; 1.5 x 10~10 and 2.6 x 10~9 M for chicken; 9.0 x 1(T10 and 9.0 x 10~9 M for turtle; 3.7 x 10"11 and 1.2 x 10"10 M for frog; and 3.0 X 10"11 and 2.7 X 10"10 M for fish. PYY was thus slightly more potent than NPY in competing for [125I]PYY binding sites. In chicken brain, APP and PPP competed equally well with both PYY and NPY with K; values of 4.6 x 10"11 and 1.1 x 10"9 M, respectively.
2 3 PYY Bound, pmol/mg protein
We also compared the binding specificity of NPY receptors to that of PYY receptors. As shown in Fig. 3, NPY receptors distinguished NPY and PYY from PPs in mammalian, reptilian, and fish brain, although in avian tissues radiolabeled NPY binding was inhibited by all of the PP family peptides examined. In all cases, Scatchard plots were curvilinear (data not shown), and examination of the high affinity component showed that
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2516
Endo • 1991 Voll29'No5
EVOLUTION OF BRAIN PYY RECEPTORS human
I
20
|I
100
I
t
A
\\\JVT sv\
60
V
40
I
IA
A
1 I
\
si
80
chicken
APP
PYY
NPY
20
n
FIG. 3. Inhibition of [126I]-BoltonHunter-NPY binding to brain membrane preparations by unlabeled NPY and structurally related peptides. Membranes were obtained from human, rat, and mouse hippocampi, chicken cerebellum, and snapping turtle and yellowtail fish brains as described in Materials and Methods and were incubated with 5 pM radiolabeled NPY for 90 min at 25 C. Varying concentrations of PYY (•), NPY (O), APP (A), and PPP (A) were also present, as shown. Nonspecific binding was subtracted from total binding, and specific binding was expressed as a percentage of maximal specific binding. Each point is the mean ± SEM of triplicate determinations from a representative experiment.
I
12
f
No 11
10
9
8
Peptida. - Los CM)
10
9
8
P.phdd. - Log
Vol. 129, No. 5 Printed in U.S.A.
Brain Peptide YY Receptors: Highly Conserved Characteristics throughout Vertebrate Evolution* MINORU OKITA, AKIO INUI, YOSHIAKI HIROSUE, MASAKI MIURA, MASAHARU NAKAJIMA, AND MASATO KASUGA Second Department of Internal Medicine, Kobe University School of Medicine, Kobe, Japan
ABSTRACT. We have shown previously that peptide YY (PYY) receptors are uniquely distributed in various mammalian brains and also have identified the receptor from porcine hippocampal membranes as a protein of 50,000 mol wt. To extend these observations, both the characteristics of PYY-receptor interaction and the structure of the receptor have been examined and compared with those of its sister peptide, neuropeptide Y (NPY), in the brains of various vertebrates including mammals (human, dog, guinea pig, rat, and mouse), birds (chicken), reptiles (snapping turtle), amphibians (bullfrog), and fish (yellowtail fish). The affinities and relative potencies of PYY as well as NPY receptors for pancreatic polypeptide (PP) family peptides were about the same in all species examined except for chickens. PYY and NPY bound to both the PYY and NPY receptors with high affinities, but porcine and avian PPs did not. In chicken brain, however, PYY, NPY, porcine PP, and avian PP all bound to the receptors with high affinity. Analysis of the equilibrium binding data for PYY receptors produced curvilinear Scatchard plots in all of the species, suggesting the existence of high and
low affinity binding sites. Affinity cross-linking using disuccinimidyl suberate followed by electrophoretic analysis of ligandreceptor complexes characterized the molecular size of PYY and NPY receptors. [125I]PYY was cross-linked to a protein of 50,000 mol wt without sulfhydryl-bonded subunits on mammalian hippocampal membranes. A receptor protein with the same mol wt was identified in other brain areas, including hypothalamus and pituitary. PYY receptors in other vertebrate brains were similar in size to those of mammalian species except in chicken brain, where a receptor protein of 67,000 mol wt was observed. In addition, we also have demonstrated that the NPY receptor is a monomeric 50,000 and 55,000 mol wt protein in mammalian and fish brains, respectively. These findings indicate that brain PYY and NPY receptors in most vertebrate species from fish to man are pharmacologically and structurally similar and have been well conserved over a period of evolution of 400 million yr. The divergence of the receptors observed in chicken brain may reflect some change in their function. {Endocrinology 129: 2512-2520, 1991)
P
EPTIDE YY (PYY), named for its C-terminal and N-terminal tyrosine residues, is a 36-amino acid peptide originally isolated from porcine duodenum (1). This peptide is a member of a family of structurally related peptides which also includes pancreatic polypeptide (PP), a pancreatic hormone (2, 3), and neuropeptide Y (NPY), a neuropeptide important in both the central and peripheral nervous systems (4). Among the porcine peptides, identities of PYY sequences with those of PP and NPY are 50 and 69%, respectively. The primary structures of porcine, rat (5), and human (6) PYY are now known. The only differences are at positions 3 and 18, where the isoleucine and asparagine residues of the human peptide are replaced by the alanine and serine residues in the porcine and rat peptides. Although PYY is found in endocrine cells of the gut and is known to Received June 4,1991. Address all correspondence and requests for reprints to: Dr. Akio Inui, Second Department of Internal Medicine, Kobe University School of Medicine, Kusunoki-cho 7-5-1, Chuo-ku, Kobe 650, Japan. * This work was supported in part by Ministry of Education, Science, and Culture of Japan Grant-in-Aid for Special Project Research 01570642.
have diverse effects on gastrointestinal function (7, 8), recent studies have revealed small quantities of PYYlike material in mammalian (9,10) and lower vertebrate (11) brains. PYY-like immunoreactivity has a topographically unique distribution and does not exhibit any overlap with other known peptide distribution, including that of NPY, suggesting a possible anatomical basis for the effects of PYY on brain function. In fact, central administration of PYY influences pituitary hormone release, thermoregulation, feeding behavior, and learning (1214). A necessary prerequisite to the biological action of peptides is an interaction with specific receptors. PYY and NPY receptors have been characterized in central and peripheral tissues and in a variety of neuronal cell lines (15-24). Furthermore, recent studies have revealed two distinct subtypes of the receptors for these peptides (21, 23, 25). Using porcine hippocampal membranes, we have demonstrated previously that PYY and NPY but not PP could bind to both the PYY and NPY receptors with great specificity (17,19). Affinity cross-linking studies also showed both brain receptors to be proteins of
2512
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 27 November 2015. at 16:00 For personal use only. No other uses without permission. . All rights reserved.
EVOLUTION OF BRAIN PYY RECEPTORS 50,000 mol wt with no subunit structures (19). However, it remains unclear whether these receptors are conserved in man or in more primitive vertebrates. No systematic attempt has yet been made to investigate the evolution of these receptors. The objective of the present study is to extend our observations in the brains of various vertebrate species from fish to man in order to understand the evolution of the receptors for PP family peptides.
Materials and Methods Reagents Synthetic porcine PYY, porcine NPY, and avian PP (APP) were purchased from Peninsula Laboratories (Belmont, CA); highly purified porcine PP (PPP) from Novo (Bagsvaerdt, Denmark); and carrier-free Na[126I] and [125I]-Bolton-HunterNPY from Amersham (Tokyo, Japan). Chloramine-T, bacitracin, phenylmethylsulfonyl fluoride (PMSF), BSA fraction V, and 2-mercaptoethanol (2ME) were obtained from Wako Pure Chemical Industries (Osaka, Japan); Sephadex G-50 superfine from Pharmacia Fine Chemicals (Tokyo, Japan); disuccinimidyl suberate (DSS) from Pierce Chemicals (Rockford, IL); mol wt standards for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) from Bio-Rad (Tokyo, Japan); and Kodak X-Omat AR films from Eastman Kodak (Rochester, NY). All other chemicals were of analytical grade. Preparation of brain membranes Human brain was obtained at postmortem from a male patient with no clinical manifestation of neurological diseases. Hartley guinea pigs, Sprague-Dawley rats, and C57BL/6 mice obtained from Japan SLC Co. Ltd. (Shizuoka, Japan) were decapitated, and their brains were rapidly removed. Brains were frozen, and hippocampi were dissected. Porcine brains were obtained from a local slaughterhouse, frozen, and dissected into hippocampi, amygdalas, caudate nuclei, and pituitary glands. Adult mongrel dogs were anesthetized with sodium thiamylal and perfused with 0.9% saline containing aprotinin. Brains were removed immediately after killing, frozen rapidly, and dissected into hippocampi, hypothalami, and pituitary glands. Chicken cerebellum and whole brains of snapping turtles, bullfrogs, and yellowtail fish were obtained soon after decapitation and frozen until used. Whole brains were used to obtain enough tissue for these species. Brain membranes were prepared as previously described (17). Briefly, brain tissues were homogenized in 5 vol ice-cold 234 mM sucrose with a motor-driven Teflon-glass homogenizer (Yamamoto Factory, Kyoto, Japan). Homogenates were centrifuged at 700 X g for 10 min to remove cellular and nuclear debris, and the supernatants were then centrifuged at 100,000 X g for 20 min at 4 C. Pellets from this spin were washed with 25 mM Tris-HCl buffer (pH 7.4) containing 1 mM PMSF and 1 mg/ml bacitracin, centrifuged again, and resuspended by homogenization in the same buffer. Membrane protein concentration was determined by the method of Bradford (26), using bovine immunoglobulin G as a standard.
2513
Receptor binding studies Synthetic porcine PYY was radioiodinated to a specific activity of 60-80 fiC'i/ng by a modification of the chloramine-T method and purified by chromatography on Sephadex G-50 superfine as described elsewhere (27). Standard incubation buffer consisted of 25 mM Tris-HCl (pH 7.4), 10 mM MgCl2) 1 mM PMSF, 1 mg/ml bacitracin, and 5 mg/ml BSA. Brain membranes at 200 /xg protein/ml were incubated in a final vol of 0.5 ml incubation buffer with 40 pM [125I]PYY for 180 min at 25 C. Binding of [126I]-Bolton-Hunter-NPY (SA, 500 /xCi/ jig) was performed similarly, except that brain membranes were incubated with 5 pM radioligand for 90 min at 25 C. Bound and free peptides were separated by centrifugation at 10,000 X g for 3 min at 4 C. Specific binding was calculated as the difference in radioactivity bound in the presence and absence of 1 /iM unlabeled PYY or NPY. Degradation of radiolabeled peptides was assessed by precipitation with trichloroacetic acid and was routinely less than 5% at the termination of incubation. Displacement data were analyzed by computerized Scatchard analysis (28). Affinity cross-linking studies Brain membranes at 400 ng protein/ml were incubated in a final vol of 1 ml 50 mM HEPES-KOH buffer (pH 7.6) containing 1 mM MgCl2, 1 mM PMSF, and 1 mg/ml bacitracin, with about 500 pM [125I]PYY or 60 pM[125I]-Bolton-Hunter-NPY for 120 min at 25 C. The receptor-bound radioligand was separated by centrifugation at 10,000 X g for 3 min, washed twice, and resuspended in 0.9 ml of the same buffer. DSS, freshly dissolved in dimethyl sulfoxide, was added to give a final concentration of 1 mM, and the cross-linking was allowed to proceed for 30 min on ice. The reaction was then quenched with 50 mM TrisHCl buffer (pH 7.6) containing 1 mM MgCl2,1 mM PMSF, and 1 mg/ml bacitracin. The cross-linked receptor-ligand complex was pelleted by centrifugation and solubilized with 50 ^1 10% SDS and 20 fA 20% glycerol in either the presence or absence of 2ME. SDS-PAGE was performed according to Laemmli (29), using 2-mm thick slab gels containing 10% acrylamide. The gels were fixed in 7% acetic acid. After visualizing the proteins with Coomassie brilliant blue, the gels were destained and dried. Autoradiograms were made from dried gels by exposure to Kodak X-Omat AR films at -80 C.
Results Pharmacological characterization of PYY and NPY binding to vertebrate brain membrane receptors The most important characterizing feature of PYY receptors in porcine hippocampus is their ability to distinguish PYY and NPY from other related or unrelated peptides (17, 19). In all species examined, [125I]PYY binding to brain membranes was inhibited by PYY and NPY in a concentration-dependent manner. PPs (APP and PPP), however, were essentially unable to inhibit [125I]PYY binding in any vertebrate species except birds. (Fig. 1) Scatchard analysis of the equilibrium binding data in
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2514
EVOLUTION OF BRAIN PYY RECEPTORS
Endo• 1991 Voll29«No5
Chicken
Human
10 9 8 7 Peptide. - L o g (M)
10 9 8 7 Peptide. -Log (M)
Turtle
Guinea
APP FIG. 1. Inhibition of [126I]PYY binding
to brain membrane preparations by unlabeled PYY and structurally related peptides. Membranes were obtained from human, guinea pig, rat, and mouse hippocampi, chicken cerebellum, and snapping turtle, bullfrog, and yellowtail fish brains as described in Materials and Methods and were incubated with 40 pM [126I]PYY for 180 min at 25 C. Varying concentrations of PYY (•), NPY (O), APP (A), and PPP (A) were also present, as shown. Nonspecific binding was subtracted from total binding, and specific binding was expressed as a percentage of maximal specific binding. Each point is the mean ± SEM of triplicate determinations from a representative experiment.
11
7 10 9 8 Peptide. -Log (M)
12
11
10 9 8 7 Peptide. -Log (M) Frog APP
12
11
10 9 8 7 Peptide. -Log (M)
12
11
10
T
Peptide. -Log (M)
Mouse
Fish
Peptide. -Log (M) 10
Peptide. -Log (M) 11
Fig. 1 appears in Fig. 2. Scatchard plots for PYY binding 4.8 x 10" M for turtle, 1.8 X 10" M for frog, and 1.9 X to vertebrate brain membranes were, in every case, cur10~n M for fish. Total binding capacities calculated for vilinear, suggesting the existence of high and low affinity the high affinity component in the respective species components of [125I]PYY binding. The high affinity comwere 0.064, 0.022, 0.14, 0.018, 0.24, 0.17, 0.008, and 0.009 pmol/mg protein. Inhibition constants (K;) (30) for PYY ponent of PYY binding showed a similar affinity from fish to man; dissociation constants were 4.8 X 10~n M and NPY were 1.4 X 10"11 and 2.6 X 10"11 M for human; n 10 for human, 8.4 x 10~ M for guinea pig, 2.8 X 10~ M 6.1 x 10"11 and 3.3 x 10"10 M for guinea pig; 1.8 X 10"10 for rat, 4.1 x 10~n M for mouse, 2.2 x 10~10 M for chicken, and 1.8 x 10"9 M for rat; 9.6 x 10" n and 1.9 X 10~10 M
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 27 November 2015. at 16:00 For personal use only. No other uses without permission. . All rights reserved.
EVOLUTION OF BRAIN PYY RECEPTORS U. 1 J
2515 0.30
Human
Chicken
i
IT \
0.10
c 3
o
0.05
CD
1 2 6 PYY Bound, pmol/mg protein
1 2 3 PYY Bound, pmol/mg protein 0.06 o i?
0.15
Guinea Pig
Turtle
0.04
C 3 co 0.02
FIG. 2. Scatchard analysis of [125I]PYY binding to mammalian and nonmammalian brain membranes. Displacement data from Fig. 1 were subjected to the analysis, and the bound to free ratio of PYY was plotted against bound PYY per milligram of protein.
2
10
20
30
2 10 20 30 PYY Bound, pmol/mg protein
PYY Bound, p m o l / m g protein 0.-06
0.06
Rat
Frog
£ 0. 04 3
& 0 02
1
2 " 8
PYY Bound, p m o l / m g protein
jnd/Free
0 .06
o CD
0
Mouse
1 4
1 10 20 PYY Bound, pmol/mg protein
Fish
0
^>—^ 1
«
2 " 10
20
30
PYY Bound. pmol/mg protein
for mouse; 1.5 x 10~10 and 2.6 x 10~9 M for chicken; 9.0 x 1(T10 and 9.0 x 10~9 M for turtle; 3.7 x 10"11 and 1.2 x 10"10 M for frog; and 3.0 X 10"11 and 2.7 X 10"10 M for fish. PYY was thus slightly more potent than NPY in competing for [125I]PYY binding sites. In chicken brain, APP and PPP competed equally well with both PYY and NPY with K; values of 4.6 x 10"11 and 1.1 x 10"9 M, respectively.
2 3 PYY Bound, pmol/mg protein
We also compared the binding specificity of NPY receptors to that of PYY receptors. As shown in Fig. 3, NPY receptors distinguished NPY and PYY from PPs in mammalian, reptilian, and fish brain, although in avian tissues radiolabeled NPY binding was inhibited by all of the PP family peptides examined. In all cases, Scatchard plots were curvilinear (data not shown), and examination of the high affinity component showed that
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2516
Endo • 1991 Voll29'No5
EVOLUTION OF BRAIN PYY RECEPTORS human
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FIG. 3. Inhibition of [126I]-BoltonHunter-NPY binding to brain membrane preparations by unlabeled NPY and structurally related peptides. Membranes were obtained from human, rat, and mouse hippocampi, chicken cerebellum, and snapping turtle and yellowtail fish brains as described in Materials and Methods and were incubated with 5 pM radiolabeled NPY for 90 min at 25 C. Varying concentrations of PYY (•), NPY (O), APP (A), and PPP (A) were also present, as shown. Nonspecific binding was subtracted from total binding, and specific binding was expressed as a percentage of maximal specific binding. Each point is the mean ± SEM of triplicate determinations from a representative experiment.
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