1 Evolution of the gastrointestinal endocrine system (with special reference to gastrin and CCK)

1 Evolution of the gastrointestinal endocrine system (with special reference to gastrin and CCK) ROD DIMALINE GRAHAM J. D O C K R A Y

The processes of digestion in the mammalian gastrointestinal tract depend on sophisticated control systems that coordinate secretion of digestive juices and movement of the luminal contents. The two most important control systems are the enteric nervous system and the epithelial endocrine system. They function in an integrated way and both are also involved in signalling to and from the central nervous system (CNS). At a molecular level there are many similarities between the two systems--in particular, both endocrine cells and enteric neurones frequently exert their actions through the release of small peptides; these, and the receptors at which they act, can be placed in families on the basis of shared amino acid sequences. During evolution it seems that a common pool of messenger peptides and receptors came to be used by both systems. Because neither peptides nor receptors are exclusively localized to the gut it is clear that the endocrine system of the digestive tract should be seen as one component of a much wider system, the function of which is control of cells through the regulated release of small peptides. In the present account, we shall deal in turn with the organization and evolution of cells that produce small peptides acting as diffusible extracellular messengers; general aspects of organization will be illustrated in detail by one particular group of peptides--those of the gastrin/cholecystokinin (CCK) family, as studies of this group are presently attracting considerable attention. Other important aspects of organization, e.g. evolution of transmembrane signalling systems, are beyond the scope of this account.

OVERVIEW

OF GUT HORMONE

EVOLUTION

Dispersed epithelial endocrine cells associated with the production of active peptides are found in the alimentary tract of all members of the vertebrates. Their peptide secretions may influence nearby cells (paracrine actions) or, after delivery in the circulation, more distant organs (hormonal actions). Cells which closely resemble the enteric endocrine cells of the vertebrates Baillidre' s Clinical Endocrinology and Metabolism--

Vol. 8, No. 1, January 1994 ISBN 0-7020-1817-1

1

Copyright © 1994, by Bailli6re Tindall All rights of reproduction in any form reserved

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R. DIMALINE AND G. J. DOCKRAY

are found in the alimentary tracts of higher invertebrates, and in protochordates which represent a stage of evolution from which the vertebrate line emerged. Whether or not the enteric endocrine cells of subvertebrate species are part of paracrine or hormonal control systems, or both, is still unclear. What is beyond doubt, however, is that control systems making use of regulatory peptide mediators are a relatively ancient feature of alimentary tract structure. The earliest origins of the regulatory peptides are a matter for speculation, but since active peptides are widely distributed in neurones of the primitive coelenterates, and since enteric endocrine cells are absent at this level of organization, it seems possible that regulatory peptides which later came to be produced in endocrine cells first emerged as neuroregulatory substances. CELLULAR EVOLUTION

Organization of the secretory pathway All cells have the capacity to secrete proteins or peptides. Cells, such as those of the enteric endocrine system, that are specialized for the secretion of small active peptides have evolved a pathway that allows storage of the secretory peptide and release on appropriate stimulation, the so-called regulated pathway. The characteristic feature of this pathway is the segregation of those peptides destined for regulated secretion from others which are released continuously by an unregulated mechanism using the so-called constitutive pathway. In some primitive organisms, e.g. yeast, the constitutive pathway is the only route for secretion, and even messenger substances, e.g. mating factors, take this pathway, but in all major animal groups, peptides or proteins functioning as extracellular messengers in the endocrine or nervous systems follow the regulated secretory pathway. The active peptides destined for release are formed from precursors translated from m R N A at the rough endoplasmic reticulum and then transported via the Golgi complex to secretory granules (Pfeffer and Rothman, 1987). Regulatory peptide precursors can be correctly sorted to secretory granules by foreign cells, so that secretory cells appear to share a common type of sorting mechanism that is able to correctly identify putative secretory peptides (Kizer and Tropsha, 1991). The nature of the secretory granule targeting signal is still unclear, and consequently its evolution remains poorly understood (Rothman, 1987; Kizer and Tropsha, 1991).

The secretory granule and its contents The secretory granules of the regulated pathway of release exhibit a characteristic spectrum of features. The granule interior is acidic, hyperosmolar, has a high concentration of nucleotides (predominantly ATP), and aside from the primary secretory product generally contains chromogranin or related soluble acidic proteins and peptide processing enzymes; typically these granules may also concentrate amines (Payne, 1989).

EVOLUTION OF GUT HORMONES

3

The acidic interior is maintained by a protein translocating ATPase; vesicles acidified in this manner have been described in animals, protozoans, algae, yeasts, moulds and higher plants (Homewod et al, 1972; Lin et al, 1977; Kakinuma et al, 1981; Bowman, 1983). The evolution of this kind of secretory granule ATPase has been speculatively linked to mitochondrial FoFz ATPase and the proton ATPases of aerobic bacteria (Ramos and Kaback, 1977). The acidic environment of the secretory granule may have an important role in enhancing the granule-stabilizing properties of ATP and the chromogranins, but in addition it may be important for the optimal action of prohormone convertases (see below). The amine and nucleotide concentrating ability of mammalian secretory granules may also be linked to the electrochemical gradient of protons. While the capacity to segregate proteins or peptides into such granules was probably an early event in their evolution, the appearance of a proton pump must have been of decisive importance because it allowed the development of a mechanism providing energy for the concentration of nucleotides and amines as well. The nearly ubiquitous distribution of chromogranins or related molecules has meant that these proteins provide useful markers for physiological or pathophysiological work on regulated secretion from endocrine cells. The function of these proteins is less clear. One view maintains that chromogranins and related molecules play a role in the condensation of material in the terminal Golgi and therefore in the segregation of peptides into secretory cells; in a related context these proteins might facilitate stabilization of the secretory granule interior in amine producing cells (FischerColbrie et al, 1987: Simon and Aunis, 1989). An alternative view is that chromogranins are the precursors of active peptides. Chromogranin A (CGA) is processed to fragments that have biological activity, including pancreastatin which inhibits secretion of several cell types including the pancreatic [3 cell (Tatemoto et al, 1986), chromostatin which inhibits secretion from bovine adrenal chromaffin cells (Galindo et al, 1991) and vasostatin which may regulate blood flow (Helle et al, 1990). There are substantial tissue and species differences in the processing of CGA, and there may be other as yet unrecognized active fragments produced from these proteins. The two views are not incompatible, and it is conceivable for example that the early functions of chromogranins during evolution were associated with secretory granule organization and that later their cleavage products became active peptides in their own right. Either way, it is interesting that chromogranin-like material has been identified in the protozoan Paramecium tetraulia, localized in the trichocysts, which are homologous with secretory granules, suggesting that these proteins are a phylogenetically ancient feature of granule organization (Peterson et al, 1987).

Processing Almost without exception peptides destined for the regulated pathway of secretion are synthesized first as higher molecular weight precursors that undergo various biosynthetic modifications including cleavage, carboxyterminal amidation, phosphorylation, sulphation or glycosylation to generate

4

R. DIMALINE AND G. J. DOCKRAY

the mature biologically active peptides. These steps are executed during passage through the Golgi compartments and after sequestration in secretory granules. The spectrum of peptides contained within a secretory granule depends on the particular peptide-encoding gene expressed, the processing enzyme genes coexpressed, and the proton ATPase and other transporters expressed and segregated to the secretory granule membrane. The synthesis of all protein and peptide precursors is associated with a single common class of ribosomes, irrespective of the final destination of the product. These molecules are almost exclusively synthesized as larger precursors that are generally inactive, so that some form of endoproteolytic cleavage is necessary for the production of active products. The most common type of cleavage is associated with pairs of basic residues. Good progress has recently been made in the characterization of the endopeptidases. The prototype for this class of enzyme is the yeast mating factor endopeptidase encoded by the Kex2 gene (Julius et al, 1984; Mizuno et al, 1988, 1989; Fuller et al, 1989b). Several mammalian endoproteases (the peptide converrases, or PCs) related to the yeast enzyme have now been cloned. The family also includes the bacterial enzyme subtilisin, indicating that it has been very well conserved during evolution. The main mammalian enzymes are PC1 (also known as PC3), PC2 and PC4 (Seidah et al, 1990, 1991; Smeekens and Steiner,1990; Smeekens et al, 1991; Nakayama et al, 1992); they generally cleave at pairs of basic residues, and in model systems are able to convert precursors such as prosomatostatin into biologically active forms. PC1 and PC2 are found in endocrine cells and neurones, but another mammalian member of the family, furin, which is located in the Golgi, is widely distributed in many different cell types (Roebroek et al, 1986; Fuller et al, 1989a; van den Ouweland et al, 1990). Thus although the PC family has become associated with proteolytic cleavage of secretory peptides throughout the eukaryotes, it is only a subgroup of enzymes that have become exclusively involved with regulatory peptide processing--probably in the specialized domain of the secretory granule. Most processing enzymes characterized so far, notably the mammalian Kex2-1ike peptide convertases, have an alkaline pH optimum and so would be relatively inactive within the secretory granule. However, one member of the family, PC2 has the crucial substitution of an Asp residue at position 310, compared with Asn in the analogous position of the other convertases. This Asn residue is important in the catalytic mechanism of subtilisin-like proteases, and substitution with Asp reduces catalytic efficiency at the alkaline pH optimum. One possibility is that the substitution of Asp in PC2 serves to restrict the enzymic activity to an acidic pH (such as found in the secretory granule) by requiring protonation of the residue for full activity. Peptide convertases homologous with PC2 and PC3 have been characterized from the amphibian Xenopus (Braks et al, 1992), and from hydra (Chan et al, 1992), respectively, and a cDNA encoding a unique Kex2-1ike protease (dKLIP-1) was recently cloned from Drosophila melanogaster (Hayflick et al, 1992). In addition to the PC group of enzymes there are probably others that are responsible for different types of proteolytic cleavage, e.g. at single basic

EVOLUTION OF GUT HORMONES

5

residues, and adjacent to proline residues. In some submammalian systems, cleavage by other enzymes such as dipeptidyl aminopeptidase may be dominant. Other post-translational events such as amidation, sulphation and phosphorylation will be considered in detail below.

MOLECULAR EVOLUTION Evolutionary strategies Hormone action is a bimolecular event involving a hormone ligand and its receptor. During evolution natural selection works on both receptor and ligand and for this reason a consideration of the evolutionary process is best made in cases where the structure of the genes, or at least the cDNAs, for both receptor and ligand are known (Table 1). Both peptide and receptor are encoded by genes in which separate exons encode the functionally important domains. The genes are assembled in an essentially mosaic pattern so that scope exists for differential splicing of exons, and duplication of exons with subsequent divergence; this capacity for variation is likely to Table 1. Gut peptides and their receptors.* Family

Representative gut peptides characterized by sequencing or cloning

Preferred receptors characterized by cloning

Gastrin/CCK

Gastrin CCK

Gastrin CCK-A and CCK-B

VIP

VIP PHI Secretin Glucagon Pituitary adenylyl cyclase activating polypeptide Gastric inhibitory polypeptide

VIP VIP? Secretin

Tachykinins

Substance P Substance K Neurokinin (NK)B

NK1 NK2 NK3

Opioids

Met- and Leu-enkephalin Dynorphin

Pancreatic polypeptide

Pancreatic polypeptide Neuropeptide Y (NPY) Peptide YY

NPY1 and NPY2

Somatostatin

Somatostatin

Somatostatin

Gastrin releasing peptide (GRP)

GRP Neuromedin B

GRP

* The major groups of gut peptides and their receptors that have been characterized by cloning and sequencing are listed. Note that in many instances, related peptides occur outside the gut, e.g. in the CNS or in amphibian skin.

6

R. DIMALINE AND G. J• DOCKRAY

have been important during evolution in accounting for the diversity of related products within families of peptides, or receptors. The pattern of organization of peptide precursors varies considerably. Both the progastrin and proCCK genes seem to encode precursors containing a single copy of the active molecule (Yoo et al, 1982; Deschenes et al, 1984) and can therefore be considered as simple or monofunctional precursors. In contrast, proenkephalin encodes four identical copies of the biologically active Met-enkephalin pentapeptide sequence, one of the related Leu-enkephalin sequence and one each of Met-enkephalin-Arg-Phe and Met-enkephalin-Arg-Gly-Leu (Comb et al, 1982; Gubler et al, 1982; Noda et al, 1982); the C-terminally extended variants have subtly different biological properties from the opioid pentapeptide. In invertebrates, peptide precursors may encode many tens of copies of the active product; this form of amplification plainly ensures the production of many molecules of transmitter from a single molecule of precursor• This might aid fast synaptic transmission, and so represent a counterpart of signalling systems utilizing small molecules, e.g. amino acids, acetylcholine (ACh), in mammalian systems. Gastrointestinal neurotransmltters are discussed in detail in Chapter 3. Certain other peptide precursors contain multiple copies of similar but not identical peptide sequences, for example in addition to glucagon, proglucagon encodes two further glucagon-like peptides (Bell et al, 1983) and proVIP contains vasoactive intestinal peptide (VIP) and the related peptide-histidine-isoleucine (PHI) (Itoh et al, 1983). In this case the different copies are clearly related, but in proopiomelanocortin (POMC) two functionally distinct peptides are produced from the same precursor: adrenocorticotrophic hormone and ~-endorphin (Nakanishi et at, 1979)• The molecular evolution of receptors is probably similar in principle to that of their ligands. The situation has recently become much clearer with the cloning of genes encoding a wide variety of receptors (see also Chapter 4). One gene superfamily that is relevant in this context encodes receptors for molecules as diverse as peptides such as CCK and the tachykinins, and non-peptides, such as ACh and noradrenaline. While it appears that there might have been many different families of receptor ligands, there are many fewer receptor families. The major family for small peptides has seven putative transmembrane domains and is G-protein linked (Probst et al, 1992)• Presumably, as new ligands appeared, they encountered variants of an existing pool of receptor molecules• The earliest origins of the receptor are uncertain, but bacteriorhodopsin is a member of this family (Unwin and Henderson, 1975; Engelman et al, 1980; Henderson et al, 1990), as is a chemoattractant receptor of Dyctostelium discoideum (Klein et al, 1988). Several yeast pheromone receptors have been described, that contain seven putative transmembrane domains (Hagen et al, 1986; Marsh and Herskowitz, 1988), but they have little amino acid sequence homology,with other members of the family so their evolutionary relationship remains unclear• The essential cellular and molecular mechanisms required for extracellular communication must therefore be extremely ancient, and the gut endocrine system of mammals should be seen as just one application to which this machinery has been adapted• •

.

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•

EVOLUTION OF GUT HORMONES

7

The gastrin/CCK family: a paradigm for hormone evolution

The gastrin/CCK family is a particularly useful illustration of the principle of hormone evolution because mammalian and submammalian peptide sequences are known, at least one submammalian gene sequence has been determined, the pharmacology of the receptors is quite well known and mammalian receptors have been cloned. Specific aspects of gastrin/CCK physiology are explained in Chapters 5 and 6. Amino acid sequences

Mammalian gastrin Gastrin was originally isolated from porcine antrum as a pair of heptadecapeptides that differed in the presence or absence of a sulphate group on the tyrosine residue in the sixth position from the C-terminus (Gregory and Tracy, 1964; Kenner and Shepherd, 1968). The biological activity of the molecule, notably for stimulation of gastric acid secretion (Figure 1), is contained in the C-terminal tetrapeptide Trp-Met-AspPheNH2, although potency is enhanced by amino-terminal extensions.

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Figure 1. Schematic representation of the major physiological roles of gastrin and CCK. Gastrin is released into the circulation from G cells of the gastric antral mucosa in response to chemical or mechanical stimulation of the lumen, or by neuronal stimulation. Its primary physiological function is to stimulate secretion of acid from parietal cells (P) and pepsinogen from zymogenic cells (Z); it may also have atrophic function on the corpus mucosa (in some species, e.g. birds, pepsinogen and acid are secreted by a common cell type). CCK is released from endocrine cells in duodenum/jejunum in response to luminal stimulation by the protein products of digestion and by fat. It stimulates pancreatic enzyme secretion and gall bladder contraction and activates a vagal afferent pathway whose efferent loop mediates gastric relaxation by VIP and nitric oxide-containing neurones.

8

R. D I M A L I N E A N D G. J. DOCKRAY

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EVOLUTION OF GUT HORMONES

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Gastrins subsequently isolated from other mammalian species had only minor, conservative substitutions (or in the case of guinea-pig, a deletion) that were outside the biologically active carboxy-terminal region (Agarwal et al, 1968, 1969a, 1969b; Gregory, 1979; Reeve et al, 1981; Bonato et al, 1986a, 1986b; Shinomura et al, 1987, 1990; Jiang et al, 1988) (Figure 2).

Mammalian cholecystokinin The active C-terminal tetrapeptide amide of gastrin is shared by CCK (Mutt and Jorpes, 1968, 1971), the hormonal stimulant of gall bladder contraction and pancreatic enzyme secretion (Figure 2). CCK contains tyrosine in the seventh position from the C-terminus, rather than the sixth position as in gastrin, and this residue must be sulphated for significant bioactivity. The position and sulphation state of the tyrosine moiety thus determines the spectrum of biological activity of the two hormones. In all species so far investigated, the structure of the C-terminal octapeptide of CCK is identical.

Birds and reptiles The first submammalian representative of the gastrin/CCK family to be isolated from the gastrointestinal tract was chicken gastrin (Dimaline et al, 1986). This molecule was isolated from a narrow ring of tissue between the avian gizzard and duodenum--the counterpart of the mammalian antrum. It shares the C-terminal tetrapeptide amide of gastrin/CCK and has a sulphated tyrosine residue in the seventh position from the C-terminus like CCK, rather than in the sixth position like gastrin; elsewhere there is little similarity between the avian and mammalian peptides (Dimaline et al, 1986) (Figure 2). However, despite this CCK-like chemistry, the chicken peptide is virtually inactive in contracting gall bladder or in stimulating pancreatic enzyme secretion in birds or mammals, but is a potent stimulant of gastric acid secretion in both orders. Hence the avian molecule is a true gastrin (Dimaline et al, 1986; Dimaline and Lee, 1990). This apparently anomalous pattern of biological activity is probably attributable to the presence of a proline residue adjacent to the sulphated tyrosine, rather than the methionine residue usually present in mammalian CCK. The proline residue may act sterically to alter the spacing of the sulphated tyrosine from the amidated C-terminus. It is not entirely clear if the proline substitution alone can explain the phenomenon; in chicken gastrin, proline is followed by aspartic acid rather than the glycine residue seen in mammalian CCK. It is conceivable that the relatively rigid aspartyl conformation enhances the steric effects of the proline. An authentic CCK, identical to the mammalian octapeptide, has since been isolated from chicken brain (Fan et al, 1987). It seems, then, that birds and mammals have used different strategies to develop distinct molecules for the stimulation of acid secretion that are devoid of cholecystokinetic or pancreozymic activities. In mammals this has been achieved by moving the position of tyrosine in the primary sequence, closer to the amidated C-terminus: in birds, the side chain of the tyrosine is

10

R. DIMALINE AND G. J. DOCKRAY

effectively shifted relative to the C-terminus by the steric effects of a proline residue, without alteration of its primary sequence position. A representative of the gastrin/CCK family containing proline at position 6 from the C-terminus was recently isolated and sequenced from the antrum of a reptile, Pseudomysscripta (Johnsen and Rehfeld, 1992). In this case the glycine residue of mammalian CCK (position 5 from the C-terminus) was present, but because the biological properties of this molecule remain unexplored it is not yet possible to designate it as a gastrin or a CCK. In an earlier study, partially purified antral extracts from another reptile, Crocodylus niloticus, had chromatographic and biological properties similar to those of chicken gastrin (Dimaline et al, 1982). If the two reptilian molecules share the C-terminal structure Tyr-Pro-Gly-Trp-Met-Asp-PheNH2, then a proline substitution alone seems able to convert a CCK-like pattern of bioactivity to a gastrin-like pattern. An authentic CCK-like sequence has not yet been reported in reptiles. The cDNA and gene sequences encoding chicken gastrin have recently been elucidated, permitting an examination of the entire peptide precursor (Wu et al, 1992) (Figure 2). This has proved to be of particular interest in the case of the region flanking the biological active C-terminus of the mature peptide. In all known mammalian and avian precursors, the region immediately following the residue that becomes the C-terminal amide in the mature peptide, has the sequence Gly-Arg-Arg. It is now well established that following cleavage of the two basic residues, the glycine is converted to an amide group by sequential reactions involving the mono-oxygenase and lyase domains of a bifunctional amidating enzyme. Following this important cleavage and amidation signal is the tripeptide sequence Ser-Ala-Glu, which again is conserved in all known mammalian precursors and in the avian progastrin. This sequence is a specific example of the general sequence Ser/Thr-X-Glu/Asp, which is a consensus sequence for phosphorylation of serine or threonine by 'physiological' casein kinase (Figure 2). In several mammalian species, phosphorylation of this serine residue has been demonstrated in both intact precursor, and in the C-terminal flanking peptide that is generated stoichiometrically with mature gastrin following cleavage and amidation (Dockray and Varro, 1993). It seems possible that phosphorylation may act as a flag for events leading to cleavage and amidation, or perhaps determination of the destination of the mature peptide. The major biologically active form of chicken gastrin is a 36 residue peptide (cG36) which, like mammalian G34, contains a pair of consecutive basic residues. Unlike mammalian G34, however, there is no evidence for tryptic cleavage of cG36 in chicken antrum. Instead, two smaller peptides have been found that correspond to residues 7-36 and 11-36 of the larger form (Dimaline et al, 1986). Conceivably these forms could be generated by three and five cycles, respectively, of dipeptidyl aminopeptidase activity. Such processing events have been described for other submammalian peptide precursors such as procaerulein (Richter et al, 1986), promellitin (Kreil et al, 1980) and the c~-mating factor of yeast (Julius et al, 1983). Examination of the sequence flanking the amino terminus of cG36 within chicken progastrin reveals another pair of basic residues, cleavage at which would produce

11

EVOLUTION OF GUT HORMONES

a peptide of 53 residues (Wu et al, 1992), and such a molecule has recently been identified in chicken antrum (Bjornskov et al, 1992).

Amphibia A decapeptide, caerulein, isolated from the skin of several amphibian species differs from CCK by a single amino acid residue within the Cterminal octapeptide (Anastasi et al, 1968). The biological properties of CCK and caerulein are virtually identical. The similar C-terminal structures of gastrin and CCK and their overlapping spectra of biological activity led to the suggestion that they may have arisen by duplication of a common ancestral gene (Dockray, 1977). Comparison of the cDNA sequences encoding gastrin and CCK and prediction of the precursor secondary structures have provided evidence to suggest such an origin (Deschenes et al, 1985b). Early immunochemical studies suggested that caerulein was present in brain and gut, as well as in the skin of amphibia, and it was suggested that this molecule might represent the ancestor of gastrin and CCK (Larsson and Rehfeld, 1977). Subsequently, however, it became clear that authentic CCK was present in amphibian brain, while caerulein was not (Dimaline, 1983); moreover, elucidation of gene sequences has revealed little similarity in the organization of the genes encoding gastrin and CCK on the one hand (Kato et al, 1983a; Ito et al, 1984; Wiborg et al, 1984; Deschenes et al, 1985a), and caerulein on the other (Vlasak et al, 1987) (Figure 3). It seems then, that 1

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Figure 3. Schematic representation of the genes encoding cholecystokinin (Deschenes et al, 1985a), gastrin (Wiborg et al, 1984) and caerulein (Vlasak et al, 1987). Introns (boxes) are numbered. Stippled boxes represent coding regions, solid boxes indicate regions encoding the peptide sequence Gly-Trp-Met-Asp-Phe-Gly, which is processed to become the biologically active C-terminal pentapeptide amide of gastrin/CCK/caerulein. Four caerulein genes (types I-IV) have been described that contain one, two, three or four copies of the peptide; exons 8-10 are absent in the type III gene illustrated (Vlasak et al, 1987).

caerulein and gastrin/CCK provide an example of convergent evolution. In the upper gastrointestinal tract of an amphibian, Rana catesbeiana (Johnsen and Rehfeld, 1992), a typical mammalian CCK-like molecule has been characterized. Taken together with the finding of a mammalian CCK8-tike peptide in Xenopus brain (Dimaline, 1983), it seems possible that amphibia do not possess a separate gastrin.

12

R. DIMALINE AND G. J. DOCKRAY

Protochordates and lower vertebrates Definitive sequences of gastrin/CCK-like peptides have so far not been obtained from fish, although material with chromatographic and immunochemical properties similar to CCK has been reported in the brain and gut of the cyclostomes Lampreta fluviatilis and L. tridentata (Holmquist et al, 1979); the occurrence of gastrin-like peptides in lower vertebrates is controversial (Fritsch et al, 1982). However, immunochemical studies on the neural ganglion of the protochordate Ciona intestinalis provided evidence for a gastrin-like peptide (Thorndyke and Dockray, 1986). This idea was confirmed with the isolation from C. intestinalis (Johnsen and Rehfeld, 1990) of an octapeptide, cionin, that has the common C-terminal amidated pentapeptide of gastrin/CCK and has tyrosine sulphate in the sixth position from the C-terminus like mammalian gastrin. Interestingly, tyrosine sulphate is also present in the seventh position from the C-terminus so that the molecule foreshadows structural features of both gastrin and CCK. While it is tempting to identify cionin as the ancestral molecule of gastrin and CCK, this is almost certainly an oversimplification; on present evidence it seems unlikely that birds or reptiles utilized a molecule with tyrosine sulphate in the sixth position from the C-terminus.

Invertebrates Several peptides having the general C-terminal sequence Tyr-Gly-X-MetX-Phe-NH2 have been identified in invertebrates (Nachman et al, 1986; Nichols et al, 1988). However, their link to the gastrin/CCK family is tenuous; indeed it could equally be argued that they belong to the insect invertebrate peptide family having the general sequence Phe-Met-Arg-PheNH2 (see Figure 2). Nothing is known of their biological actions. Patterns of gene expression In classes that possess a distinct gastrin, e.g. mammals, birds, reptiles, the gene is expressed mainly in the antral part of the stomach. In contrast the CCK gene is expressed in both brain and gut of all vertebrate classes. In protochordates and invertebrates the situation is less clear. Thus while cionin was isolated from the neural ganglion ('brain') of C. intestinalis (Johnsen and Rehfeld, 1990), immunohistochemical studies have reported gastrin/CCK-like material in gastrointestinal tract (Fritsch et al, 1982), and CCK-like biological activity was demonstrated in the gut of another ascidian, Styela davis (Thorndyke and Bevis, 1983). The dual expression of gastrin/CCK-like peptides in neurones and endocrine cells therefore seems to be an ancient feature. RECEPTOR EVOLUTION Early binding studies Evidence for the status of cholecystokinin as a major neuropeptide in the

EVOLUTION OF GUT HORMONES

13

central nervous system was presented in 1976 (Dockray, 1976), and in 1978 CCK8 was isolated and sequenced from mammalian brain (Dockray et al, 1978). Two years later, Innis and Snyder demonstrated that the binding site for radiolabelled CCK in mammalian brain was different from that of the pancreatic CCK receptor (Innis and Snyder, 1980). It subsequently became clear that the characteristics of the CCK brain binding site were similar to those of the receptor for gastrin in gastric corpus mucosa. However, these early studies were hampered by the lack of specific high affinity receptor antagonists for either gastrin or CCK. Nevertheless, using radiolabelled natural ligands, and their synthetic analogues, it was possible to demonstrate that the mammalian pancreatic CCK receptor is highly selective for sulphated forms of CCK. In contrast both brain and stomach receptors distinguish poorly between sulphated and unsulphated forms of CCK, gastrin, or the common C-terminal tetrapeptide amide. The two classes of CCK receptors are now known as A and B. CCK-A receptors are found predominantly in the alimentary tract, but are also found in discrete areas of the CNS, e.g. the rat interpeduncular nucleus and the nucleus tractus solitarius (Hill et al, 1987). What is less clear is whether the CCK-B receptors that are widely distributed in the CNS are identical to the peripheral gastrin receptor. Studies using radiolabelled agonists suggest that the CCK-B and gastrin receptors might differ (Mennozi et al, 1989; Sutcliffe et al, 1990; Patel and Spraggs, 1992), while binding studies using radiolabelled highly selective antagonists have emphasized the similarities between the two receptor types (Hunter et al, 1993). Brain binding sites for mammalian CCK have also been demonstrated in lower vertebrates, including representatives of Agnatha and Osteichthyes (Williams et al, 1985). Studies on CCK receptors in an amphibian, Rana catesbeiana, indicated that, in contrast to mammals, neither pancreatic nor brain receptors were able to distinguish between gastrin or CCK (Vigna et al, 1984). In an extension of these studies (Vigna et al, 1986) it was demonstrated that in representatives of three classes of ectothermic vertebrates (Amphibia, Chondrichthyes and Reptilia) the specificities of brain and pancreatic CCK receptors are similar in that they do not distinguish between gastrin and CCK, although they do favour the sulphated variants of each peptide, and interact only weakly with the tetrapeptide amide. In contrast, avian pancreatic and brain CCK receptors display specificity patterns similar to those of their mammalian counterparts. These findings led to the hypothesis that in ectotherms the CCK receptor might represent a primitive molecule from which the more distinct pancreatic and brain receptors in endotherms have evolved. It has further been speculated that evolution of a receptor tolerating sulphated tyrosine in either position 6 or 7 from the C-terminus to a receptor requiring tyrosine sulphate in position 7 may have been coincident with or subsequent to the evolution of gastrin from an ancestral CCK-like molecule (Vigna et al, 1986). The brain CCK receptor in higher vertebrates also plainly differs from that in lower vertebrates in that it does not require a sulphated tyrosine and has a high affinity binding domain for the C-terminal tetrapeptide amide region of gastrin/CCK (Figure 4).

14

R. DIMALINE AND G. J. DOCKRAY

LOWER VERTEBRATES

HIGHER VERTEBRATES

~

~

ptides: CCK-like eptor CCK-SO4 =

[

BRAIN

cificity: Gastrin-SO~]

city: G a s t r i n - ~

GUT

ptides:

CCK

eptor (~ cificity:

CCK = I (NS=s~Gastrin I

Fptides:

CCK Gastrin

/

[Receptor

~ CCK = Gastrin

I I

~

ity:

(NS_-SO,l I 0 ooK-so, ] >>>G~

Figure 4. Specificity of gastrin/CCK-receptor types in higher and lower vertebrates. The single lower vertebrate receptor requires tyrosine sulphate in the sixth or seventh position from the C-terminus of the agonist (i.e. sulphated gastrin or CCK-like molecules). The higher vertebrate gastrin/CCK-B receptor will tolerate sulphated or non-sulphated (NS) forms of gastrin and CCK, while the CCK-A receptor is highly specific for sulphated CCK.

Development of high affinity selective antagonists The development, over the last few years, of specific high affinity receptor antagonists for gastrin and CCK has facilitated the classification of these receptors, as well as permitting more incisive studies on the physiological actions of this peptide family. The earliest reported gastrin/CCK receptor antagonists were cyclic nucleotides such as dibutyryl cyclic GMP or amino acid derivatives such as proglumide; these were weak antagonists, however, and generally did not distinguish between CCK-A and CCK-B receptors (see, for example, Freidinger, 1989). Subsequently, more potent, selective antagonists were devised, based variously on derivatives of proglumide, the benzodiazepine nucleus, and peptide or pseudopeptide analogues of gastrin/ CCK (Freidinger, 1989). One particularly useful benzodiazepine derivative, L 364,718 (MK 329, devazepide), is potent (effective at 0.1-1.0 mg/kg, in vivo) and highly selective for the CCK-A receptor (Chang and Lotti, 1986). A related compound, L 365,260, is selective for CCK-B receptors (Block et al, 1989), but is neither as potent nor as selective as devazepide. Recently, however, a potent and highly selective CCK-B receptor antagonist, C1988 (PD 134308), has become available (Hughes et al, 1990); the compound is a non-peptide analogue of CCK. Although highly selective CCK antagonists such as those described above have enabled unequivocal distinction between brain and alimentary receptors, some anomalous species differences have come to light. Thus in dog, the benzodiazepine antagonist L 365,260, which in rodents is selective for the gastrin/CCK-B receptor, has a tenfold lower affinity for the parietal cell gastrin receptor than does

EVOLUTION OF GUT HORMONES

15

devazepide, which in rodents is highly specific for the CCK-A receptor. How useful these receptor antagonists will be in evolutionary studies remains to be established, but there is already evidence to show that devazepide is not an effective antagonist at the avian CCK-A receptor, despite the fact that this receptor shows a typical mammalian pattern of agonist specificity (Campbell et al, 1991). Structural studies

Recent cloning and expression studies have elucidated the cDNA sequences encoding several members of the gastrin/CCK receptor family. To date, three receptor sequences have been elucidated, the gastrin receptor from the canine parietal cell (Kopin et al, 1992), the CCK-A receptor from rat pancreas (Wank et al, 1992a) and the CCK-B receptor from rat brain (Wank et al, 1992b). Moreover, it has been demonstrated by polymerase chain reaction (PCR) cloning that the CCK-A receptor seen in highly localized regions of rat CNS (see above) is identical to the receptor found in pancreas (Wank et al, 1992b). Indirect evidence that the CCK-B and gastrin receptors may be identical comes from studies that show CCK-B-type receptors on the AR42-J rat pancreatic acinar carcinoma cell line to be identical to the brain receptor (Wank et al, 1992b). Unequivocal demonstration of this point will require comparison of receptor sequences derived from brain and parietal cells in the same species. All three gastrin/CCK receptor amino acid sequences contain seven putative transmembrane domains, suggesting that they are members of the guanine nucleotide binding-coupled receptor superfamily (Figure 5). There are significant homologies with the [3-adrenergic receptor subfamily, and also similarities to the receptors for the tachykinins, gastrin releasing peptide and neuropeptide Y (Kopin et al, 1992; Probst et al, 1992; Wank et al, 1992a, 1992b). The CCK-A and CCK-B receptors in rat share almost 50% homology, largely within the putative transmembrane domains and in the first and second intracellular loops. The least conserved regions are the amino terminus and the third intracellular loop; the latter differs in A and B receptors in both length and composition (Figure 5). In the other seven transmembrane domain receptors, the third intracellular loop is known to be important in G-protein coupling (Dixon et al, 1987; O'Dowd et al, 1988). Other pointers to membership of the G-protein binding family are conserved cysteine residues in the first and second extracellular loops, which may be important for tertiary structure stabilization as demonstrated for rhodopsin, muscarinic and [3-adrenergic receptors, and a cysteine residue in the cytoplasmic tail which may have a membrane anchoring function (Ovchinnikov et al, 1988; Hulme et al, 1990). There is also about 50% homology between the dog gastrin receptor and the rat CCK-A receptor; the chief regions of diversity are similar to those described above for the CCK-A and CCK-B receptors (Figure 5). In contrast, the dog gastrin receptor and rat CCK-B receptor are highly homologous, and in particular the second extracellular loop and third intracellular loops differ at only 30% of amino acid residues. These small differences may

16

R. DIMALINE AND G. J. DOCKRAY

Gastrin ~

~

COOH

~ CCK-A

(Rat)

~

~

~

NHz

o

COOH NH2

cc.

Figure 5. Schematic representation of cloned and sequenced gastrin/CCK receptors. Upper panel, dog parietal cell gastrin receptor; regions homologous with the CCK-A receptor are indicated by filled circles. Centre panel, rat CCK-A receptor. Lower panel, rat CCK-B receptor; regions homologous with the CCK-A receptor are indicated by filled circles. Hatched boxes represent the cell membrane, the amino-terminal tail is extracellular, the carboxyterminal tail is cytoplasmic. Putative transmembrane domains were assigned by Kyte-Doolittle analysis (Wank et al, 1992a, 1992b) or the Klein, Kanehisa, DeLisi algorithm (Kopin et al, 1992). Filled triangles represent consensus sequences for N-linked glycosylation. Stars represent potential protein kinase A, protein kinase C or casein kinase II phosphorylation sites.

17

EVOLUTION OF GUT HORMONES

well reflect species variation and strongly suggest identical extracellular ligand specificity and intracellular G-protein coupling specificity of these two receptors. The gastrin/CCK receptors so far sequenced comprise a single subunit protein, and in at least one case successful screening of clones depended upon this property (Kopin et al, 1992). With the availability of cDNA sequences encoding both CCK-A and gastrin/CCK-B receptors it should be possible to apply PCR cloning strategies to determine amino acid sequences of submammalian representatives of this family. Of particular value in elucidating evolutionary pathways will be the sequence of the ectothermic CCK receptor, and of the receptor for the invertebrate gastrin/CCK-like peptide, cionin. CONTROL MECHANISMS Although paracrine, hormonal and neuronal control mechanisms can be recognized in all major vertebrate groups, the manner in which the available pool of messengers is utilized within these systems may differ markedly, even in the control of closely similar biological functions. This point is well illustrated by the way that birds and mammals have evolved distinct functions for the same three regulatory peptides, gastrin, gastrin releasing peptide (GRP) and somatostatin, in the control of gastric acid secretion (Figure 6).

MAMMALS

_l_

SOM • H+ ®

A$~SOM

J[-

BIRDS

Acid Secreting Mucosa

Gastrin

Gizza.~ /, ,,,

Secreting Muccsa

Figure 6. Schematic representation of the upper gastrointestinal tract of birds and mammals, and hormonal regulators of acid secretion. Acid-secreting (corpus) and gastrin-secreting (antrum) mucosas are contiguous in mammals. In birds, the acid-secreting proventriculus is separated from the antrum by the muscular gizzard. Established mechanisms of control are indicated by solid arrows; proposed mechanisms are shown by broken arrows (see text). SOM, somatostatin; GAS, gastrin; GRP, gastrin releasing peptide.

18

R. DIMALINE AND G. J. DOCKRAY

In mammals, gastrin from the gastric antral mucosa is released into the bloodstream by luminal chemical and mechanical stimulation, and elicits acid secretion from the parietal cells of the gastric corpus mucosa. In the mammalian stomach, gastrin releasing peptide (GRP) is localized exclusively in neurones, and, as its name implies, stimulates release of antral gastrin. Gastrin release is inhibited by somatostatin, which is in turn released by luminal acid. Somatostatin is located in endocrine cells of the antral mucosa that are closely associated with gastrin cells and which influence the gastrin cells by local or paracrine secretion. The secretion of antral gastrin and somatostatin is reciprocally related, as is the abundance of the messenger RNAs that encode them; when luminal acidity is high, somatostatin biosynthesis and secretion is high, while gastrin biosynthesis and secretion is low (see, for example, Debas, 1987; Yamada, 1987). If acid is removed from the lumen by treatment with a proton pump inhibitor such as omeprazole, somatostatin biosynthesis and secretion are depressed, while gastrin biosynthesis and secretion are elevated (Brand and Stone, 1988; Karnik et al, 1989; Wu et al, 1990; Dimaline et al, 1991). Somatostatin is also present in endocrine cells of the corpus mucosa, but the activity of these cells appears not to be regulated by luminal acidity (Sandvik et al, 1993). Instead it seems that calcitonin gene related peptide (CGRP) in primary afferent neurones is at least one factor regulating corpus somatostatin (Sandvik et al, 1993). In birds, too, gastrin and somatostatin are present in endocrine cells of the antral mucosa. However, while antral gastrin biosynthesis is markedly elevated when birds are made achlorhydric, there is no reciprocal change in antral somatostatin. Instead, somatostatin is depressed in the acid secreting mucosa (proventriculus) suggesting that in birds somatostatin may regulate antral gastrin hormonally rather than in a paracrine manner, since the antrum is physically separated from the proventriculus (Wu et al, 1992). The function of, and mechanisms controlling avian antral somatostatin remain to be elucidated. Moreover, unlike its neuronal mammalian counterpart, avian GRP is located in endocrine cells of the proventriculus, and there is evidence to suggest that it is released by chemical and mechanical stimulation of the proventriculus mucosa and acts hormonally (Campbell et al, 1991). Plainly the basic elements of the control systems are retained in birds and mammals, but what has changed is the particular mode of delivery adopted for different regulatory peptides. SUMMARY

The evolution of gut endocrine cells can be seen to have depended in the first instance on the expression of genes encoding regulatory peptides in cells that had evolved the regulated pathway of secretion. It seems probable that the endocrine cells made use of molecules and mechanisms that first emerged in early nervous systems. However, by the start of the vertebrate line of evolution, most of the major families of gut hormones were already found in association with endocrine cells. A single common class of receptor with

EVOLUTION OF GUT HORMONES

19

s e v e n t r a n s m e m b r a n e d o m a i n s a n d acting via association with G - p r o t e i n s t r a n s d u c e s m a n y ( p e r h a p s all) gut p e p t i d e actions. T h e d u p l i c a t i o n a n d d i v e r g e n c e of r e c e p t o r s a n d p e p t i d e s c a n n o w b e traced, in o u t l i n e at least, for gastrin a n d C C K in v e r t e b r a t e s . E v e n in p h y l o g e n e t i c a l l y similar groups such as birds a n d m a m m a l s , q u i t e different m o l e c u l a r a p p r o a c h e s have b e e n a p p l i e d to solving the s a m e physiological p r o b l e m . E v o l u t i o n of the m o d e r n g a s t r o i n t e s t i n a l c o n t r o l system e v i d e n t l y d e p e n d e d in this case b o t h o n m o l e c u l a r e v o l u t i o n of p e p t i d e s ahd r e c e p t o r s a n d o n cells expressing the genes e n c o d i n g t h e m .

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