Parathyroid hormone-receptor interactions

Parathyroid hormone-receptor interactions

vide direction for design 01 clinicall? useful antagonists for calcium disorders related to PTH excess or malignancyassociated hypercalcemia. Parathy...

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vide direction for design 01 clinicall? useful antagonists for calcium disorders related to PTH excess or malignancyassociated hypercalcemia.

Parathyroid Interactions Michael

P. Caulfield

Hormone-Receptor and Michael

Rosenblatt

Identification of sites within the antagonist peptide of parathyroid hormone (PTH) that are “tolerant” of a wide range of amino acid substitutions has led to the design of new PTH antagonists. These antagonists have increased potency because of substitution, at appropriate sites, of amino acids that increase the interaction of the ligand with its receptor but do not cause signal transduction. Similar modifications in the parathyroid hormone-related protein (PTHrP) antagonist led to antagonists with increased potency. Further, the partial agonism of this analog could be removed by exchange of residues between PTH and PTHrP.

Structure-function studies of hormones have provided endocrinologists with insights into the nature of hormone-receptor interactions and the mechanism of action of a number of hormones. Manipulation of structure can identify features critical for bioactivity within a hormone molecule and may indirectly indicate presumed sites of complementarity within the receptor, which form the basis of hormone-receptor interactions. Analysis of conformational effects on hormonal activity can also elucidate the conformation favored by receptors. Until recently, only examination of the role of hormonal structure (accomplished in great detail for some hormones) has been possible in endocrine systems. Now, the cloning and expression of a few receptors is enabling experiments to be conducted in which receptor structure is modified by recombinant DNA techniques (site-directed mutagenesis) and the consequences for hormone-binding or signal transduction are analyzed (Dixon et al. 1988; Lefkowitz and Caron 1988). For parathyroid hormone (PTH), at this time, only the traditional hormoneoriented structure-activity studies have Michael P. Caulfield and Michael Rosenblatt are at Department of Biological Research and Molecular Biology, Merck Sharp and Dohme Research Laboratories, West Point, PA 19486, USA.

164

been performed. The receptor, although partially characterized biochemically (Brennan and Levine 1987; Goldring et al. 1984; Karpf et al. 1987; Shigeno et al. 1988). has yet to be cloned and expressed in functional form. In this review, we summarize nearly two decades of research on PTH-receptor interactions. We have delineated largely separable functional domains within the hormone, responsible for receptor binding and receptor activation. Much has been learned, both directly and indirectly, about the chemistry and biology of the PTH system in an effort to generate potent and selective antagonists. These investigations have led to the creation of potent hormone antagonists that are effective in vivo. Recently, a tumor-secreted calciummobilizing hormone, termed parathyroid hormone-related protein (PTHrP), has been characterized (Suva et al. 1987; Mangin et al. 1988; Thiede et al. 1988). Since this hormone exerts many, if not all, of its actions through PTH receptors, it provides an additional and entirely new structural framework for integration into the design of PTH analogs. Synthesis and biological evaluation of molecular chimeras of PTH and PTHrP already have identified subtle structural features, which, when introduced into an “antagonist” backbone, can activate receptors. These studies promise to pro-

The early structure-function studies by Potts et al. (1971) demonstrated that full biological activity for the conventional indices of mineral ion flux of PTH resides within the N-terminal 34 amino acids. An increase in agonist potency was observed when the C-terminal Phe residue was substituted by a Tyr-amide (Potts et al. 1982). Substitution of methionines at positions 8 and 18 with norleutines, an almost isosteric replacement, was well tolerated. The combination of these two modifications produced an oxidation-stable peptide that could be radiolabeled with iodine while retaining full biological activity (Rosenblatt et al. 1976). The resulting PTH radioligand has been extensively used by several laboratories in PTH radioreceptor binding assays that have played a key role in evaluating potential antagonists. Studies by Goltzman et al. (1975) showed that removal of the two N-terminal amino acids resulted in a dramatic decrease in agonist properties. The 3-34 fragment was identified as the first antagonist of PTH action. Subsequently, a 3-34 analog, [Nle8f’8,Tyr34]bPTH (3-34)NH,, was shown to be a more potent antagonist of PTH both in binding assays and PTH-stimulated adenylate cyclase assays (Rosenblatt et al. 1977) and is still routinely used in vitro. Formal kinetic analyses revealed that the analog was a true competitive antagonist that competed for receptor occupancy with PTH on an equimolar basis. In the late 197Os, when this analog was first prepared, it represented the most potent hormone antagonist designed for any peptide hormonal system. The promise of this antagonist led the group working on this project to dedicate their efforts to a large-scale synthesis of the analog in order to enable in vivo evaluation of its properties. When examined in vivo, in several animal models, the analog was found to be a weak partial agonist (Rosenblatt et al. 1986). Although disappointing, these results were not surprising since, in several other hormonal systems, the progression from antagonists effective in vitro to those effective in vivo has been impeded by the retention of weak agonist activity seen only when these compounds are used in high doses in vivo.

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The reasons for these developments were not clear, but the pathway toward a solution to the problem was to identify the principal binding sequence of the hormone. Analysis of the binding properties of multiple fragments of the l-34 region delineated a critical binding domain within the region 25-34. The region is remote (in terms of linear sequence) from the N-terminal domain critical for full activation of receptors. These studies also demonstrated diminished, but acceptable, binding for the 7-34 sequence. Once again, large-scale synthesis was undertaken, this time of [Tyr341bPTH(7-34)NH,. This peptide lacked agonist properties in vivo and was shown to be an antagonist for the major indices of PTH action in vivo (Horiuchi et al. 1983; Doppelt et al. 1986) including the calcemic and phosphaturic responses . Whereas it is an effective antagonist, [Tyr34]bPTH(7-34)NH2 is not a suitable candidate for clinical use, as its potency is too low; molar excesses of antagonist over agonist of 200: 1 are required for complete inhibition of PTH action (Horiuchi et al. 1983). Recent work in our laboratory, based on conformationally oriented substitutions, has now led to PTH antagonists with substantially increased potencies compared with [Tyr34]bPTH(7-34)NH,. Michael Chorev and Ruth Nutt have focused on biologically “tolerant” sites within the molecule, i.e., positions that can accept a wide latitude of substitutions with little or no effect on bioactivity. Identification of such sites makes possible the incorporation of substitutions, based on conformational predictions, designed to optimize the interaction of antagonist with receptor without restoring agonist properties or diminishing the specificity of receptor interaction. This can be accomplished by either introducing new moieties that add binding elements not present in the nativesequence agonist or introducing substitutions that stabilize the antagonist in a conformation favored by the receptor. Initial structure-function studies have focused on one particular struetural motif-the turn. In the l-34 region of PTH, two regions have been identified as having the potential to form turns. One turn, a y-turn, has been proposed to occur in the region 20-24

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Binding’ 4, (nW

Analog [Nle8~1s,Tyr34]bPTH(7-34)NH, [Nle8,1s,Leu23,Tyr34]bPTH(7-34)NH~

150 * 10 2260 * 910

[Nles,18,N-MePhe23,Tyr34]bPTH (7-34)NH2 [Nle8~‘s,N-MeGlu22,Tyr34]bPTH

> 30,000

(7-34)NH, [Nles~‘8,D-Trp23,Tyr34]bPTH(7-34)NH2

8000 t 76,000

1500

? 15,000

Adenylate cyclase’ 4 fnW 1550 * 330 > 7100 NTb 1 39,800 96,000

2 16,000

* Assays were performed as described (Goldman et al. 1988). Values represent means +- SEM b NT, not tested.

(Bundi et al. 1978). The other turn, a p-turn, is predicted by the Chou-Fasman algorithm of protein secondary structure to occur around Gly’*. The putative y-turn was examined because earlier studies (Potts et al. 1982) had indicated that this region was biologically “tolerant.” The y-turn structure was proposed to be stabilized by the interaction of the side groups of Va12’and Trp23. Studies were undertaken using amino acid substitutions known to stabilize yturns and others that interfere with the interaction of Va12’ and Trp23 (Table 1). Substitutions of amino acids that stabilize y-turns, N-methyl Glu for Glu”, and N-methyl Phe for Trp23 resulted in analogs with diminished potencies. Substitutions that do not contribute to stabilizing y-turns, Leu or D-Trp for Trp23, also resulted in less potent analogs. Hence, conclusions regarding the conformation around position 23 could not be made. Whatever the structure around position 23, it is now clear that this region is not as tolerant as we had supposed. Rather, modification generally results in decreased potency. Studies examining Gly’* were more informative and rewarding. A p-turn is predicted in this region and again single amino acid substitutions, designed to stabilize or destabilize the turn, were incorporated into [Tyr34]bPTH(7-34)NH,. Replacement of Gly’* with amino acids such as sarcosine or proline, which stabilize a p-turn, decreased the potency of the antagonists (Table 2). However, replacement with amino acids that do not break a helix were well tolerated. Contrary to the Chou-Fasman prediction, the preferred structure around po-

sition 12 appears to be helical. Ultimately, these findings led to the design of the most potent PTH antagonist yet reported (Goldman et al. 1988), an analog with D-Trp incorporated at position 12. This substitution increases potency lo- to 30-fold compared with [Tyr341bPTH(7-34)NH,. Similarly, (Y and p n-naphthylalanine substitutions have resulted in more potent antagonists. Thus, substitution groups at position

of large hydrophobic 12 increases receptor

affinity, presumably by providing favorable interactions with a site in the receptor that is not normally utilized by native-sequence agonists. The antagonist properties of [n-Trp’2,Tyr341bPTH (7-34)NH, are currently being examined in vivo. One of the most exciting and important developments in this field has been the characterization of a new hormone, the tumor-secreted PTHrP. Compelling evidence has been obtained documenting the direct interaction of this hormone with PTH receptors in vitro and in vivo (Horiuchi et al. 1987; Kemp et al. 1987; Juppner et al. 1988; Fukayama et al. 1988; Stewart et al. 1988; Yates et al. 1988; Rabbani et al. 1988; Thompson et al. 1988). Although there is a welldefined short N-terminal domain of striking homology with PTH, the remainder of the PTHrP structure is nonhomologous (Figure 1). Nevertheless, PTHrP has a biological profile similar to PTH and appears responsible for producing the hypercalcemic syndrome associated with many human malignancies. This finding is unique in the field of endocrinology. While there are other examples of one hormone acting (be-

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165

7-34

Bindinga Analog

Adenylate

* * ? 2

10 40 30 10 40

500 ? 50r 110 * lo+

90 10 10 1

80t 260 110 120 470

[Nles.‘8,Tyr34]bPTH(7-34)NH2 [Nle8~‘s,n-a-Na112,Tyr34]bPTH(7-34)NH, [Nle8~‘8,n-P-Na112,Tyr34]bPTH(7-34)NH,

cyclasea K fnM)

4, fnW 840 840 410 610 1400 2510 540 740 70

t 70 ? 180 r 70 * 120 * 670 + 730 2 140 * 110 *lo

150 +- 10 10 + 1

1550 + 330 350 + 70

10 * 2

140 -+ 40

a Assays were performed as described (Goldman et al. 1988). Values are means t SEM

cause of inherent structural similarity) on another hormone’s receptor, such as growth hormone and prolactin, vasopressin and oxytocin, insulin and insulin-like growth factor 1 (for references, see Rosenblatt 1986), a dramatic decline in receptor affinity (usually lOO-fold or more) usually is evident as the move across receptor systems is made. In this case, PTH and PTHrP are virtually identical in affinity for and efficacy at PTH receptors. This structure-function experiment of nature has important implications for design of antagonists of both PTH and PTHrP. Roberta McKee and co-workers have found that, if the region of primary

sequence homology, the N-terminal 13 residues, is removed from both hormones, the remaining 14-34 fragments still compete with comparable affinity for PTH (or PTH/PTHrP) receptors. These experiments indicate that the “binding” domain regions of both hormones must present comparable structural, and probably conformational, features to receptors despite lack of sequence identity. Conceptually similar findings were also obtained by NUSSbaum and co-workers (Abou-Samra et al. 1989). Another revealing finding has been obtained through structure-function studies of PTHrP. It was obvious that the

Figure 1. Sequence comparison of the N-terminal Asterisks indicate amino acid homology.

34 amino

acids of hPTH and hPTHrP

15

10 hPTHrP

Ala Val Ser Glu His Gin Leu Leu His Asp Lys Gly Lys Ser Ile Gln Asp

hPTH

Ser Val Ser Glu Ile

+:: 96,::::: :: **

20

::* * :: **

* ,b“'

*** **:>

Gln Leu MetHis Asn Leu Gly Lys His Leu Asn Ser 25

30

His Leu Ile Ala Glu Ile His Thr Ala

hHPTHrP

Leu Arg Arg Arg Phe Phe Leu His *** :L:::"

hPTH

Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His Asn Phe

166

:: ::i .c

fragment

of PTHrP might be a po-

tent antagonist. Indeed, the fragment has binding affinity and inhibitory potency comparable to [Tyr34]bPTH (7-34)NH, (McKee et al. 1988). Unlike its PTH counterpart, however, the 7--34 fragment of PTHrP is a weak partial agonist both in vitro and in vivo. As with PTH, PTHrP has a glycine at residue 12, and substitution of n-Trp at this position results in an antagonist with a sixfold increase in potency and a reduction in partial agonist activity compared with PTHrP(7-34)NH,. Other structure-function studies based upon a comparison of hybrid molecules of the PTH and PTHrP sequences have been undertaken. Two nonhomologous amino acids in the N-terminal domains have been substituted for each other. In the case of [A~p’~,Lys”,Tyr”~]bPTH(7-34)NH,, the Aspi and Lys” of PTHrP have been substituted into the PTH sequence to replace the native-sequence Asn’O and Leu”. These modihcations alone have conferred partial agonist properties. By contrast, the corresponding PTHrP peptide, [Asn’O, Leu”lPTHrP(7-34)NH,, is devoid of partial agonism. This kind of experiment demonstrates that biological properties such as agonism can be inserted in “cassette form” into an appropriate “receptor-binding” sequence (see Figure 2). Furthermore, these studies indicate that the “activation” domain can be extended farther into the molecule, to positions 10-12, than previously postulated by us. A successful structure-activity program requires as much emphasis on the biological assays as on the design of peptide analogs. It can be greatly facilitated by the development of in vitro assays that accurately and reliably detect weak agonism since such a finding might preclude the usefulness of a particular antagonist in vivo. Efforts in our laboratory are now under way to establish such an in vitro assay. Along similar lines, the goals of analog design need to be kept closely in mind as a program proceeds. Our goal has been to design antagonists of PTH (and now PTHrP) that might be useful in treatment of human disease. Recently, Roberta McKee has found evidence of important differences between rat and human PTH receptors that could have adversely affected our antagonist program had we relied exclusively on rat-based systems. Based on comparison

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TEM JamuqlFebruayv

through the use of subtly altered analogs of PTH. Cloning of the PTH receptor will provide direct information regarding

PTH 10

10

11

7+,,.4

qAsp.t----,,.----J~

Exchange 10

the nature of the PTH receptor as well as insights into the mechanism of action

Antagonist, partial agonist

Antagonist, no partial agonism

7+

11

of PTH.

residues

11

10

References

11

Asp, Lys +/24 Antagonist, partial agonist

Figure 2. Effect of exchange

Antagonist, no partial agonism

of amino acids at positions

10 and 11 between

PTH and PTHrP-

(7-34)NH, on partial agonist properties. Amino acids at positions 10 and 11 are enclosed within either an oval or a rectangle for PTH and PTHrP peptides, respectively. across species, our design directions have led to antagonists that are 50 to 100 times more potent in human bone than rat bone systems. Obviously, such information requires us to shift our emphasis for a wide range of bioassays to human-derived tissues. Although promising directions have emerged from each of the several approaches to design of analogs of the hormones described above, we still have very little information regarding the other fundamental component of hormone-receptor bimolecular interaction, the receptor. Although the PTH receptor has been partially characterized, its structural elucidation and recombinant DNA expression remain to be achieved. Several groups are now hard at work in efforts to purify and eventu-

ally clone the PTH receptor. The impetus for these efforts is the notion that the availability of the cloned PTH receptor will provide new insights into the nature of PTH-receptor binding and signal transduction. Structure-function studies based on manipulation of the receptor itself then could be undertaken to identify critical elements within the receptor required for interaction with either agonists or antagonists. This information could guide future analog design. Finally, the entire issue of the existence of one versus several PTH receptor subtypes (Figure 3) and the association of PTH with multiple transmembrane signalling mechanisms (such as CAMP, polyphosphoinositols, and intracellular calcium fluxes) now can be pharmacologically addressed only

Figure 3. Diagrammatic representation of possible mechanisms by which PTH can stimulate alternate second messengers in addition to adenylate cyclase; a one-receptor system interacting with different G proteins vs multiple subtypes of receptors interacting with discrete G proteins.

CAMP

TEA4 January/Febmary

Abou-Samra A-B, Uneno S, Jueppner H, et al. Non-homologous sequences of parathyroid hormone and parathyroid hormone related peptide bind to a common receptor on ROS 1712.8 cells. Endocrinology 1989; 125:2215. Brennan DP, Levine MA: Characterization of soluble and particulate parathyroid hormone receptor using a biotinylated bioactive hormone analog. J Biol Chem 1987; 262: 14,795. Bundi A, Andreatta RH, Wuthrich K: Characterisation of a local structure in the synthetic parathyroid hormone fragment l-34 by ‘H nuclear-magnetic-resonance techniques. Eur J Biochem 1978; 91:201. Dixon RAF, Strader CD, Sigal IS: Structure and function of G-protein coupled receptors. Annu Rep Med Chem 1988; 23:221. Doppelt SH, Neer RM, Nussbaum SR, Federice P, Potts JT Jr, Rosenblatt M: Inhibition of the in vivo parathyroid hormone-mediated calcemic response in rats by a synthetic hormone antagonist. Proc Nat1 Acad Sci USA 1986; 8317557. Fukayama S, Bosma TJ, Goad DL, Voelkel EF, Tashjian AH Jr: Human parathyroid hormone (PTH)-related protein and human PTH: comparative biological activities on human bone cells and bone resorption. Endocrinology 1988; 123:2841. Goldman ME, McKee RL, Caulfield MP, et al: A new highly potent parathyroid hormone antagonist: [n-Trp”, Tyr34]bPTH-(7-34) NH,. Endocrinology 1988; 123:2597.

IP, + DAG

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Goldring SR, Tyler GA, Krane SM, Potts JT Jr, Rosenblatt M: Photoaffinity labeling of parathyroid hormones receptors: comparison of receptors across species and target tissues and after desensitization to hormone. Biochemistry 1984; 23:498. Goltzman D, Peytremann A, Callahan E, Tregear GW, Potts JT Jr: Analysis of the requirements for parathyroid hormone action in renal membranes with the use of inhibiting analogues. J Biol Chem 1975; 250:3199. Horiuchi N, Holick MF, Potts JT Jr, Rosenblatt M: A parathyroid hormone inhibitor in vivo: design and biological evaluation of a hormone analog. Science 1983; 220: 1053. Horiuchi N, Caulfield MP, Fisher JE, et al: Similarity of synthetic peptide from human tumor to parathyroid hormone in vivo and in vitro. Science 1987; 238:1566. Juppner H, Abou-Samra A-B, Uneno S, Gu W-X, Potts Jr JT, Segre GV: The parathyroid hormone-like peptide associated with humoral hypercalcemia of malignancy and parathyroid hormone bind to the same receptor on the plasma membrane of ROS 17/2.8 cells. J Biol Chem 1988; 263:8557. Karpf DB, Arnaud CD, King K, et al.: The canine renal parathyroid hormone receptor is a glycoprotein: characterization and partial purification. Biochemistry 1987; 26:7825. Kemp BE, Moseley JM, Rodda Parathyroid hormone-related malignancy: active synthetic Science 1987; 238:1568.

CP, et al.: protein of fragments.

Lefkowitz RJ, Caron MG: Adrenergic receptors: models for the study of receptors coupled to guanine nucleotide regulatory proteins. J Biol Chem 1988; 263:4993. Mangin M, Webb AC, DreyerBE,et al.: Identification of a cDNA encoding a parathyroid hormone-like peptide from a human tumor associated with humoral hypercalcemia of malignancy. Proc Nat1 Acad Sci USA 1988; 85:597. McKee RL, Goldman ME, Caulfield MP, et al.: The 7-34-fragment of human hypercalcemia factor is a partial agonist/antagonist for parathyroid hormone-stimulated CAMP production.Endocrinology 1988; 122:3008. Potts JT Jr, Tregear GW, Keutmann HT. et al.: Synthesis of a biologically active N-terminal tetratriacontapeptide of parathyroid hormone. Proc Nat1 Acad Sci USA 1971; 68~63. Potts JT Jr, Kronenberg HM, Rosenblatt M: Parathyroid hormone: chemistry, biosynthesis, and mode of action. Adv Prot Chem 1982; 35:323. Rabbani SA, Mitchell J, Roy DR, Hendy GN, Goltzman D: Influence of the amino-terminus on in vitro and in vivo biological activity of synthetic parathyroid hormone-like peptides of malignancy. Endocrinology 1988; 123:2709.

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Rosenblatt M: Peptide hormone antagonists that are effective in vivo. N Engl J Med 1986; 315:1004. Rosenblatt M, Goltzman D, Keutmann HT, Tregear GW, Potts JT Jr: Chemical and biological properties of synthetic, sulfur-free analogues of parathyroid hormone. J Biol Chem 1976; 251:159. Rosenblatt M, Callahan EN, Mahaffey JE, Pont A, Potts JT Jr: Parathyroid hormone inhibitors: design, synthesis, and biological evaluation of hormone analogues. J Biol Chem 1977; 252~5847. Shigeno C, Hiraki Y, Westerberg DP, Potts JT Jr, Segre GV: Parathyroid hormone receptors are plasma membrane glycoproteins with asparagine-linked oligosaccharides. J Biol Chem 1988; 263:3872. Stewart AF, Mangin M, Wu T, et al.: Synthetic human parathyroid hormone-like protein stimulates bone resorption and causes hypercalcemia in rats. J Clin Invest 1988; 81:596.

Suva

LJ, Winslow

GA, Wettenhall

REH,

et

al.: A parathyroid hormone-related protein implicated in malignant hypercalcemia. cloning and expression. Science 1987; 237:893. Thiede MA, Strewler GJ, Nissenson RA. Rosenblatt M, Rodan GA: Human renal carcinoma expresses two messages encoding a parathyroid hormone-like peptide: evidence for the alternative splicing of a single-copy gene. Proc Nat1 Acad Sci USA 1988; 85:4605. Thompson DD, Seedor JG, Fisher JE, Rosenblatt M, Rodan GA: Direct action of the parathyroid hormone-like human hypercalcemic factor on bone. Proc Nat1 Acad Sci USA 1988; 85:5673. Yates AP, Gutierrez GE, Smolens P, et al.: Effects of a synthetic peptide of a parathyroid hormone-related protein on calcium homeostasis, renal tubular calcium reabsorption, and bone metabolism in vivo and in vitro in rodents. J Clin Invest 1988; 81:932. TEM

Peptide YY and Neuropeptide Y in the Gut Availability, Biological Actions, and Receptors Marc Laburthe

Intensive research on the actions of peptide YY(PYY) and neuropeptide Y (NPY) on the gut has been stimulated by findings of potent anti-secretory effects in small intestine and the discovery of their common receptor in this tissue. There is evidence that the hormone PYY and the neurotransmitter NPY are involved in the regulation of fluid and electrolyte secretion, motility, and blood flow in the intestine. Intestinal PYYINPY receptors may have pharmacological value for the treatment of diarrhea.

Peptide

YY

(PYY),

neuropeptide

Y

(NPY), and pancreatic polypeptide (PP) are three members of a family of regulatory peptides that exhibit considerable sequence homology. PP was first isolated in 1975 as a by-product of insulin

Marc Laburthe is at the Department of Differentiation and Neuroendocrinology of Digestive Cells, Institut National de la SantC et de la Recherche MCdicale (INSERM U178), BLtiment INSERM, 16 Avenue Paul Vailiant Couturier, 94807 Villejuif Cedex, France.

purification from the pancreas (Kimmel et al. 1975). PYY and NPY were isolated in 1982 with a chemical assay that detected the presence of COOH-terminalamide in peptides (Tatemoto et al. 1982). In pigs, there are 18 sequence identities (50% homology) between PP and PYY or NPY and 25 sequence identities (70% homology) between PYY and NPY (Clover et al. 1985 and Figure 1). PYY and NPY are highly conserved peptides during evolution, with very similar sequences in most mammalian species.

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