Guanylin Peptides and Cgmp 1329 ISSN 0100-879X

Brazilian Journal of Medical and Biological Research (1999) 32: 1329-1336 Guanylin peptides and cGMP 1329 ISSN 0100-879X

Guanylin peptides: cyclic GMP signaling mechanisms

L.R. Forte1,2, 1Harry S. Truman Veterans’ Hospital, Departments of R.H. Freeman3, 2Pharmacology, 3Physiology and 4Pathology and Anatomical Sciences, W.J. Krause4 and School of Medicine, Missouri University, Columbia, MO, USA R.M. London1,2

Abstract

Correspondence Guanylate cyclases (GC) serve in two different signaling pathways Key words L.R. Forte involving cytosolic and membrane enzymes. Membrane GCs are · Kidney Department of Pharmacology receptors for guanylin and atriopeptin peptides, two families of cGMP- · Intestine School of Medicine · regulating peptides. Three subclasses of guanylin peptides contain one Guanylate cyclase Missouri University · Chloride secretion intramolecular disulfide (lymphoguanylin), two disulfides (guanylin M-515 Medical Sciences Building · Sodium excretion Columbia, MO 65212 and uroguanylin) and three disulfides (E. coli stable toxin, ST). The - USA peptides activate membrane receptor-GCs and regulate intestinal Cl - Fax: +1-573-884-4558 and HCO3 secretion via cGMP in target enterocytes. Uroguanylin and E-mail: [email protected] ST also elicit diuretic and natriuretic responses in the kidney. GC-C is an intestinal receptor-GC for guanylin and uroguanylin, but GC-C Presented at the Meeting “NO Brazil, Basic and Clinical may not be involved in renal cGMP pathways. A novel receptor-GC Aspects of Nitric Oxide”, expressed in the opossum kidney (OK-GC) has been identified by Foz do Iguaçu, PR, Brazil, molecular cloning. OK-GC cDNAs encode receptor-GCs in renal March 10-13, 1999. tubules that are activated by guanylins. Lymphoguanylin is highly expressed in the kidney and heart where it may influence cGMP pathways. Guanylin and uroguanylin are highly expressed in intestinal mucosa to regulate intestinal salt and water transport via paracrine Received May 28, 1999 actions on GC-C. Uroguanylin and guanylin are also secreted from Accepted June 22, 1999 intestinal mucosa into plasma where uroguanylin serves as an intestinal natriuretic hormone to influence body Na+ homeostasis by endocrine mechanisms. Thus, guanylin peptides control salt and water transport in the kidney and intestine mediated by cGMP via membrane receptors with intrinsic guanylate cyclase activity.

Introduction as biologically active, 13, 14 and 15 amino acid peptides using the T84 cell cGMP bio- Guanylin, uroguanylin and lymphogua- assay (2). Lymphoguanylin was recently iden- nylin are heat-stable peptides that regulate tified using a PCR-based homology cloning the enzymatic activity of cell-surface guany- strategy to isolate cDNAs from opossum late cyclase signaling molecules. Guanylin spleen and other lymphoid tissues, which was the first endogenous peptide identified encode a 109 amino acid precursor that is and was isolated as a 15 amino acid peptide most similar in its primary structure to pre- from rat intestine that stimulates cGMP pro- prouroguanylin (3). Guanylin and uroguany- duction in T84 intestinal cells (1). Urogua- lin have two intramolecular disulfide bonds, nylin was next isolated from opossum urine but lymphoguanylin has only one disulfide

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bond. A synthetic form of lymphoguanylin protein (11,12). CFTR and GC-receptors are also activates guanylate cyclase (GC)-recep- localized together in apical plasma mem- tors in human T84 intestinal and opossum branes of target enterocytes. The first GC- kidney (OK) cells. The family of guanylin receptor for guanylin family peptides was regulatory peptides share similarities both in identified at the molecular level (i.e., GC-C) primary structures and biological activities by molecular cloning of cDNAs encoding an with the heat-stable enterotoxin (stable toxin, intestinal membrane protein that binds 125I- ST) peptides secreted by strains of enteric ST with high affinity and is activated by ST microorganisms that cause a watery form of (13). Transgenic suckling mice with dis- diarrhea similar to that seen in cholera (4). abled GC-C genes have a marked reduction Thus, bacteria-derived ST peptides are mo- in the intestinal fluid secretion response to E. lecular mimics of guanylin peptides acting to coli ST, indicating that GC-C is responsible stimulate fluid and electrolyte secretion into for a major component of the intestinal ac- the lumen of the intestine by activation of tions of guanylin peptides to enhance fluid native guanylin receptors located on the api- secretion (14,15). However, about 10% of cal surfaces of enterocytes lining the intes- specific 125I-ST binding to receptor sites on tine. Biologically active peptides in the intestinal membranes still remain functional guanylin family of proteins are found at the in GC-C-knock out (GC-C-KO) animals (15). COOH-termini of longer precursor polypep- This suggests that an additional gene or mul- tides that are secreted as biologically inac- tiple genes encoding GC-receptors for tive prohormones or protoxins (5-8). Pro- guanylin agonists exist in the mouse ge- teolytic enzymes serve as converting en- nome. zymes to activate the proguanylins or proSTs, Bacterial STs were the first peptides but these important enzymes have not been shown to activate GC-receptor signaling identified thus far. molecules in the intestine, thus causing secretory diarrhea (4). Moreover, the intestinal Physiological actions of guanylin GC targets for ST peptides in the intestine peptides were considered unique and not existing outside the intestinal epithelium until we Physiological actions of the endogenous discovered that E. coli ST also stimulates guanylin peptides include the regulation of GC-receptors found on the surface of OK intestinal fluid secretion during digestion, and potoroo kidney (PtK-2) cell lines (Fig- and neutralization of HCl in the duodenum ure 1; Ref. 16). It can be seen from one of our and of organic acids derived from enteric original experiments that these two kidney bacteria in the large intestine (9,10). Guany- cell lines have remarkable cGMP responses lins and STs stimulate the electrogenic se- to E. coli ST, but only small cGMP re- cretion of both chloride and bicarbonate an- sponses to atriopeptin-A and no detectable ions, which provides the physiological driv- responses to the nitric oxide donor, sodium ing force to accomplish fluid secretion into nitroprusside. The additional demonstration the intestinal lumen. Control of these intesti- that kidney cortex has specific GC-receptors nal functions by guanylin peptides is medi- that are activated by ST implies that this ated via intracellular cGMP through activa- class of cell surface GC-receptors plays a tion of cGMP-dependent protein kinase II much broader physiological role in the body (cG-kinase II) and/or cAMP-dependent pro- than was previously recognized (17-19). It tein kinase II (cA-kinase II) with subsequent should be emphasized that these findings phosphorylation of the cystic fibrosis trans- were made well before the first guanylin membrane conductance regulator (CFTR) peptides were isolated (1,2). Putative ST

Braz J Med Biol Res 32(11) 1999 Guanylin peptides and cGMP 1331 receptors were also found in other epithelia nylin are also found in the circulation and it of the opossum in addition to the receptors is likely that the gastrointestinal (GI) tract is localized to brush border membranes (BBM) a main source of the plasma peptides (7,33, of epithelial cells lining the intestinal tract 34). Secretion of uroguanylin from GI mu- and within renal tubules (16-19). The exist- cosa into the plasma in response to oral NaCl ence of renal, hepatic, airway and testicular may explain the prolonged increase in uri- ST receptors predicted that endogenous nary sodium excretion that occurs following ST-like peptides exist to regulate the activity a high salt meal (26,27). of the extra-intestinal as well as intestinal GC-receptors. These seminal experiments Identification of a kidney led directly to the subsequent isolation of GC-receptor for uroguanylin guanylin and uroguanylin peptides from intestine and urine, respectively (1,2). Both We sought to elucidate the primary struc- guanylin and uroguanylin are produced in ture of a membrane GC expressed in cul- the intestine, but uroguanylin is the major tured OK cells and in the opossum kidney bioactive peptide found in urine, which con- because the prior discovery of this renal GC- tains either no guanylin or very small a- receptor was a stimulus that ultimately led to mounts of the peptide (2,20-22). Uroguany- the isolation of guanylin and uroguanylin lin and ST stimulate the enzymatic activity (1,2,16-19). A PCR-based cloning strategy of renal tubular GC-receptors and increase was used to isolate 3762-bp cDNAs from the urinary excretion of sodium, potassium RNA/cDNAs expressed in OK cells and and water in both the perfused rat kidney ex opossum kidney cortex (35,36). Transfec- vivo and the mouse in vivo (16-19,23-25). tion and expression of the OK-guanylate Guanylin is less potent in the stimulation of cyclase (OK-GC) cDNA into COS and urinary Na+ and water excretion compared HEK293 cells produces a cell surface GC- to either uroguanylin or ST, but guanylin receptor of ~160 kDa size that is activated by does have marked kaliuretic activity in the uroguanylin, guanylin and E. coli ST pep- perfused rat kidney (24). Thus, uroguanylin tides. OK-GC cDNA contains an open read- has biological activity consistent with a pep- ing frame encoding a 1049 residue mature tide hormone that influences renal function by regulating the urinary excretion of so- 70000 13000 dium chloride as a physiological mechanism OK PtK-2 12000 that contributes to the maintenance of Na+ 60000 11000 balance in the body. A natriuretic peptide 50000 10000 cells) cells) such as uroguanylin was predicted to exist in 6 6 9000 the digestive system for release into the blood- 40000 8000 stream for the purpose of stimulating the 30000 7000 urinary excretion of NaCl following a salty 20000 6000

meal (26,27). Uroguanylin is a prime candi- cGMP (pmol/10 1000 500 cGMP (pmol/10 date for this intestinal natriuretic hormone 750 500 250 because it is produced at extraordinarily high 250 0 0 concentrations in the upper small intestine Basal ST ANP-A Na-NP Basal ST ANP-A Na-NP and is released following a high salt meal (28-32). Uroguanylin mRNAs are most abun- Figure 1 - Activation of surface receptors on OK and PtK-2 cells by Escherichia coli stable toxin (ST) and atriopeptin-A. Confluent cells were treated for 15 min at 37oC in DMEM- dant in the small intestine compared to HEPES-MIX with 0.5 µM ST, 0.1 µM atriopeptin-A (ANP-A) or 1 mM sodium nitroprusside guanylin mRNA levels, which peak in the (Na-NP). The data are reported as the mean of 4 experiments with opossum kidney (OK) large intestine. Uroguanylin and prourogua- cells and 2 experiments with potoroo kidney (PtK-2) cells.

Braz J Med Biol Res 32(11) 1999 1332 L.R. Forte et al.

protein belonging to the family of membrane and/or guanylin peptides that signals via GC-receptor signaling molecules. OK-GC is cGMP to regulate the urinary excretion of similar to other membrane GC proteins con- sodium, potassium and water (23-25). OK- taining NH2-terminal agonist-binding do- GC is the first kidney receptor for guanylin mains, a single membrane span and intracel- family peptides to be fully defined at the lular kinase-like and GC catalytic domains. molecular level. Prior to the identification of OK-GC is 72, 76 and 75% identical in its GC-receptor signaling molecules for E. coli overall structure compared to the intestinal ST in the OK and PtK-2 cells and in the BBM-localized GC-C receptors for guanylin opossum kidney, it was thought that ST- peptides found in rats, humans and pigs, stimulated GCs were restricted to intestinal respectively (13,37,38). The catalytic do- mucosa (16-19). Thus, identification of OK- mains of OK-GC and GC-C receptors of rat, GC in OK cells and opossum kidney opened human and porcine intestine share 92, 94 up a new field of inquiry that culminated and 95% identity, respectively. The most recently with the discoveries of guanylin, highly variable region of membrane GC- uroguanylin and lymphoguanylin peptides receptors occurs within the NH2-terminal (1-3). These peptides activate OK-GC and ligand-binding domains of these proteins. may serve as endogenous agonists for this OK-GC shares only 55-59% identity in this membrane GC-receptor. The OK cell line domain when compared to GC-C intestinal has the differentiated properties of renal receptors for guanylin peptides. The GC-C proximal tubules and receptor autoradiogra- receptors of the rat are more closely related phy experiments with 125I-ST have clearly to human and pig GC-C in the ligand-bind- shown that specific binding sites for this ing domains with these proteins sharing ~70% uroguanylin-like radioligand are found in identity in this region. Thus, OK-GC is a cells of both convoluted and straight por- distinctive renal GC-receptor that, along with tions of proximal tubules (16-19). High lev- the intestinal GC-C receptor, provides two els of 125I-ST-labeled receptors are also found distinctive molecular subtypes of GC-recep- in BBMs isolated from the kidney and intes- tors for a growing family of membrane re- tine, indicating that this GC-receptor is pref- ceptors for the guanylin peptides. erentially localized to apical plasma mem- OK-GC mRNA levels were measured in branes of both kidney and intestinal target total RNA prepared from tissues of opos- cells. It is likely that OK-GC serves as a sums by Northern and RT-PCR. A 3.8-kb physiological receptor for uroguanylin, which mRNA was detected in kidney, OK cells, is the major bioactive guanylin family pep- urinary bladder, adrenal gland, heart and tide in urine (2,20-22). However, guanylin intestine. Lower levels of OK-GC mRNA was also isolated from opossum urine indi- were detected in renal medulla compared to cating that this peptide may influence renal cortex. Tissues with lower levels of OK-GC function in vivo via cGMP (2). Active uro- mRNA are urinary bladder, adrenal gland guanylin and inactive prouroguanylin pep- and both the ventricles and atria of the heart. tides circulate in plasma, thus providing a In the GI tract, high levels of OK-GC mRNA source for the urinary forms of bioactive were measured in both the small and large uroguanylin in opossum, human and rat urine intestine. Thus, OK-GC mRNA is highly (2,20-22). The kidney also expresses uro- expressed in the kidney cortex and intestinal guanylin and guanylin mRNAs, which of- mucosa with mRNA transcripts occurring at fers the possibility that an intra-renal signal- lower levels in a number of other organs. ing pathway exists for the guanylin peptides OK-GC is a candidate for the renal tubu- as well as providing another potential source lar receptor that is activated by uroguanylin of uroguanylin in the urine (35,39). Physi-

Braz J Med Biol Res 32(11) 1999 Guanylin peptides and cGMP 1333 ologically, uroguanylin may regulate kidney ing frame within the lymphoguanylin cDNAs function via an endocrine axis linking the encode a 109 amino acid polypeptide that is intestine to the kidney and/or through an 84% identical to preprouroguanylin and 40% intra-renal paracrine mechanism. Both pos- identical to preproguanylin (7,35). At the sibilities involve the activation of OK-GC as COOH-terminus of the 109 amino acid pre- a key signaling molecule in renal tubular prolymphoguanylin is a 15 amino acid pep- target cells that possess the guanylate cy- tide that is 80% identical to uroguanylin, but clase-cGMP signaling machinery. shares only 40% identity with guanylin (Fig- ure 3). Illustrated for comparison are the Identification of lymphoguanylin structures of opossum lymphoguanylin, uroguanylin, guanylin and an E. coli ST peptide, A third member of the guanylin family of which form three different subclasses of peptides was sought since biologically ac- guanylin peptides based on the number of tive uroguanylin peptides were isolated from intramolecular disulfides found within the opossum urine (2). This inquiry culminated active peptides. A major difference within in the isolation of cDNAs encoding opossum lymphoguanylin is the tyrosine109 residue preprolymphoguanylin, a third and unique because guanylins and uroguanylins have member of the guanylin family of endogenous regulatory peptides (3). The identifica- 1 86 100 tion of lymphoguanylin stems from recent + - 3HN Guanylin SHTCEICAFAACAGC CO2 experiments showing that mRNA transcripts for guanylin and/or uroguanylin and their + 1 95 109 3HN Uroguanylin QEDCELCINVACTGC - GC-receptors are expressed broadly in tis- CO2 sues of the digestive, renal-urinary, cardio- 1 95 109 + vascular, reproductive, lymphoid-immune Lymphoguanylin QEECELCINMACTGY - 3HN CO2 and central nervous organ systems (7,35). Moreover, hybridization assays revealed the Figure 2 - Prepropeptide structures for guanylin, uroguanylin and lymphoguanylin. The existence of a novel mRNA transcript that amino acid sequences of the active peptides are shown using the single letter abbreviation hybridizes with both uroguanylin and of the amino acids within the COOH-terminal regions of each opossum polypeptide. guanylin cDNA probes, but is substantially longer than either the 1.2-kb uroguanylin or Class I 0.8-kb guanylin mRNAs (3). We identified a NSSYCCELCCNPACTGCY N third guanylin-related mRNA transcript by E. coli ST molecular cloning using a homology cloning strategy based on the PCR with cDNAs iso- Class II SCH TGE I C A FAACC A lated from opossum tissues. The deduced Guanylin polypeptide was named preprolymphoguanylin because the first cDNAs isolated were Uroguanylin QCETDVE L C I NACCG derived from several different lymphoid organs and the encoded polypeptide is similar Class III in primary structure to preprouroguanylin Lymphoguanylin QCETEME L C I NAYCG and preproguanylin (Figure 2). Sequence analyses of multiple independent cDNA Figure 3 - Comparison of the primary structures of subclasses within the guanylin family of clones reveals that the lymphoguanylin biologically active peptides. The peptide subclasses are provided from top to bottom in the order of their discovery as: Class I peptides containing 3 disulfides, class II peptides with 2 cDNA is 92.7% identical to the correspond- disulfides and class III peptides that have only 1 intramolecular disulfide bond. The single ing nucleotide sequences for preprourogua- letter abbreviation is used to designate amino acid residues in each peptide. E. coli ST, E. nylin reported previously (7). An open read- coli stable toxin.

Braz J Med Biol Res 32(11) 1999 1334 L.R. Forte et al.

cysteine residues at this position (1,2). The mRNAs for lymphoguanylin using Northern disulfide bonds formed between the first and assays in RNA from spleen, thymus and third and second to fourth cysteines in the testis. Spleen appears to have the most abun- peptide chain were thought to be required dant levels of lymphoguanylin mRNA within for biological activity of guanylin and uro- tissues of the lymphoid/immune system. RT- guanylin. The replacement of cysteine109 with PCR was used to amplify the mRNA-cDNAs the tyrosine109 residue is a novel molecular of spleen, thymus, lymph nodes, circulating change within the guanylin family of pep- white blood cells, bone marrow, cerebellum tides. Lymphoguanylin is uroguanylin-like and testis. Lymphoguanylin cDNAs were because it has two glutamate residues in its cloned and sequenced to confirm that lym- NH2-terminal domain. Opossum uroguany- phoguanylin mRNAs are expressed in thy- lin has glutamate and aspartate residues and mus, lymph nodes, circulating white blood all other uroguanylin molecules in mamma- cells, bone marrow, spleen, cerebellum, kid- lian species have acidic residues at these ney, OK cells, testis, ovary and heart of the positions (2,20,29,30,39). Shared between opossum. lymphoguanylin and uroguanylin is an inter- nal asparagine residue, which is also found Conclusion in the bacterial ST peptides. Guanylin peptides have an aromatic amino acid at this Three different guanylin regulatory pep- position (1,2,6,7,35). The third difference is tides, guanylin, uroguanylin and lymphogua- the methionine104 substitution in lymphoguanylin, together with their cognate receptors, nylin for the valine104 of uroguanylin. GC-C and OK-GC, provide signal transduc- A lymphoguanylin peptide was synthe- tion machinery for cGMP-mediated regula- sized and oxidized to form an intramolecular tion of cellular function in the kidney, intes- disulfide bond between cysteine98 and cys- tine and other epithelia. Combinatorial com- teine106, with cysteine100 protected. Synthetic plexity is achieved by mixing and matching lymphoguanylin containing a single disul- certain subtypes of GC-receptors with cell- fide stimulates cGMP production in human specific expression of one or more guanylin T84 intestinal cells, but its potency is less peptides for local control of cGMP levels in than uroguanylin or guanylin (3). All three nearby target cells. A model for this type of guanylin peptides are full agonists in the local regulation by a paracrine mechanism is stimulation of intestinal GC-C expressed in best exemplified by the guanylin-cGMP-ion human intestinal T84 cells (1-3). When the transport mechanisms that have been rigor- potencies of these peptides were examined ously demonstrated in the intestinal mucosa. in OK cells, we observed that the OK-GC An endocrine axis for uroguanylin serving receptors were also activated by lymphogua- as an intestinal natriuretic hormone involved nylin. in the maintenance of body sodium balance Lymphoguanylin mRNA transcripts of has emerged as well. The recent discovery of ~1.6 kb were detected in total RNA prepara- lymphoguanylin coupled with evidence that tions using Northern hybridization assays additional subtypes of membrane GC-recep- with a lymphoguanylin cDNA probe (3). tors exist for guanylin peptides suggest that Although lymphoguanylin cDNAs were iso- signal transduction pathways utilized by the lated first from lymphoid tissues, we were guanylin family of regulatory peptides are surprised to discover that tissues with the considerably more complicated than previ- most abundant lymphoguanylin mRNAs are ously suspected. It is likely that novel physi- the atria and ventricles of heart as well as ological actions of the guanylin regulatory kidney cortex. We also detected ~1.6 kb peptides will be discovered as more details

Braz J Med Biol Res 32(11) 1999 Guanylin peptides and cGMP 1335 are learned concerning the cellular and mo- central nervous organ systems. This will lecular mechanisms of action of these im- complement the physiological roles of portant peptide hormones. Physiological roles guanylin and uroguanylin that have been for guanylin peptides in the regulation of documented thus far in the regulation of target cell function via intracellular cGMP fluid and electrolyte transport within the in- are likely to be documented in the future for testine and kidney. the immune, reproductive, cardiovascular and

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