Guanylyl

cyclases,

a growing

family

of signal-transducing

enzymes DORIS

KOESLING,

Institut

f#{252}r Pharmakologie,

EYCKE

BOHME,

AND G#{220}NTERSCHULTZ

Freie Universtit#{227}t Berlin,

ABSTRACT Guanylyl cyclases, which catalyze the formation of the intracellular signal molecule cyclic GMP from GTP, display structural features similar to other signal-transducing enzymes such as protein tyrosinekinases and protein tyrosine-phosphatases. So far, three isoforms of mammalian membrane-bound guanylyl cyclases (CC-A, CC-B, CC-C), which are stimulated by either natriuretic peptides (CC-A, CC-B) or by the enterotoxin of Escherichia coli (CC-C), have been identified. These proteins belong to the group of receptorlinked enzymes, with different NH2-terminal extracellular receptor domains coupled to a common intracellular catalytic domain, In contrast to the membrane-bound enzymes, the heme-containing soluble guanylyl cyclase is stimulated by NO and NO-containing compounds and consists of two subunits (a1 and f3). Both subunits contain the putative catalytic domain, which is conserved in the membrane-bound guanylyl cyclases and is found twice in adenylyl cyclases. Coexpression of the a1- and /3 1-subunit is required to yield a catalytically active enzyme. Recently, another subunit of soluble guanylyl cyclase was identified and designated /32, revealing heterogeneity among the subunits of soluble guanylyl cyclase. Thus, different enzyme subunits may be expressed in a tissue-specific manner, leading to the assembly of various heterodimeric enzyme forms. The implications concerning the physiological regulation of soluble guanylyl cyclase are not known, but different mechanisms of soluble enzyme activation may be due to heterogeneity among the subunits of soluble guanylyl cyclase.-Koesling, D.; B#{246}hme,E.; Schultz, G. Guanylyl cyclases, a growing family of signal-transducing enzymes. FASEB J. 5: 2785-2791; 1991. Key Words:

transducer

transrne?nbrane signaling

peptide

hormone

IN A MULTICELLULAR ORGANISM, the communication between cells is maintained by hormones and neurotransmitters. Most hormones do not pass the cell membrane but bind to specific cell-surface receptors, which transduce the signal to the inside of the cell. This transmembrane signaling is achieved by different systems. Most receptors for hormones and neurotransmitters are known to elicit cellular responses by interacting with specific receptors coupled to effector functions by G proteins (1) (Fig. 1). The interaction between receptor, transducer (G protein), and effector is consecutive and reversible. The /3-adrenoceptor functionally coupling to the adenylyl cyclase via G probably represents the bestknown example of this type of hormone-sensitive signal transduction system. The receptors and transducers involved exhibit structural similarities, e.g., all receptors contain seven putative membrane-spanning regions, and all G proteins consist of three subunits that reveal high homology among the different isoforms; in contrast, the structures of the effectors are quite diverse, e.g., the adenylyl cyclases are

0892-6638/9110005-2785/$O1.50.

© FASEB

D-1000 Berlin 33, Germany

single subunit membrane-bound enzymes whereas the rod cGMP-phosphodiesterase is a heteromeric soluble enzyme. The second type of receptors involved in transmembrane signaling is permanently linked to the effector system; here, binding of the ligand and generation of the intracellular signal are functions of one molecule. Receptor-linked effector systems can either be ion channels or enzymes. Examples for receptors with catalytic activity are the growth factor receptor-linked protein tyrosine kinases (2) and the receptorlinked protein tyrosine phosphatases (3-5), which consist of various membrane-bound forms in which different extracellular receptor domains are linked to a common catalytic domain (see Fig. 1). Besides these membrane-bound receptorlinked forms, tyrosine kinases and phosphatases occur in cytosolic forms, which exhibit a catalytic domain homologous to that of the membrane-bound forms. Recent progress in research on guanylyl cyclases indicates that the cGMPgenerating enzymes exhibit similar structural characteristics. Although cyclic GMP, the product of the guanylyl cyclases (which plays an important role in smooth muscle relaxation, platelet aggregation, and the vision system), was discovered 20 years ago, as were cytosolic and particulate guanylyl cyclases (6), it was not until recently that the mechanisms of the physiological regulations of the membrane-bound and the cytosolic enzymes were clarified and different isoforms of guanylyl cyclases were identified (7) (Table 1). So far three forms of mammalian membrane-bound guanylyl cyclases have been identified; they show largely conserved intracellular regions but differ in the extracellular ligand-binding domain. The soluble form of guanylyl cyclases was shown to contain a region homologous to the intracellular region of the membrane-bound enzymes. Besides these structural analogies to the families of protein kinases and protein phosphatases, the guanylyl cyclases exhibit sequence homologies to the adenylyl cyclases; these homologous regions encircle putative catalytic domains.

PLASMA CYCLASES

MEMBRANE-BOUND

GUANYLYL

Regulation Membrane-bound guanylyl cyclases represent a recently discovered family of proteins whose activity is stimulated by different peptide hormones (see Table 1). The chemotactic peptide resact, which is synthesized by sea urchin eggs and causes changes in sperm motility, was shown to stimulate membrane-bound guanylyl cyclase of the sea urchin sperm (8). Atrial natriuretic (ANP)2 and related peptides, all of

‘To whom correspondence should be addressed at: Institut f#{252}r Pharmakologie, Freie Universit#{228}t Berlin, Thielallee 67-73, D-1000 Berlin 33, Germany. 2Abbreviations: ANP, atrial natriuretic peptide; SDS, sodium dodecyl sulfate; EDRF, endothelium-derived relaxing factor.

2785

om www.fasebj.org by Tulane Univ Howard-Tilton Lib (129.81.226.78) on December 02, 2018. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNumber()},

rsceptor

O-ptoteln

adenylyl cyclasa

which cause vasodilation and two main classes of natriuretic initially

NI4 ANP NH

mci C045

EGF

LAH

PDGF

rsceptOrs r.c.ptor.

P1K

PTP

Pm PIP

cyc

cyc co

cOCH

co prot#{149}in-tyroslns

prot.In-tyroslns

pho$phates

cyc

co

o

Qusnylylcycl.s.s

kin...,

Figure 1. Schematic transduction

across

representation membranes.

of two pathways The

upper

panel

used in signal represents

the

signaling system in which receptor (e.g., /3-adrenoceptor), transducer (e.g., G5), and effector (adenylyl cyclase) are localized on distinct molecules. The lower panel shows the receptor-linked membrane-bound forms of protein-tyrosine phosphatases, proteintyrosine kinases, and guanylyl cyclases and their respective cytosolic forms. Labeled boxes indicate sequence homologies. PTP, putative catalytic domain of protein-tyrosine phosphatases; PTK, putative catalytic domain of protein-tyrosine kinases; cyc, putative catalytic domain of guanylyl and adenylyl cyclases; CD 45, leukocyte common antigen; LAR, leukocyte common antigen-related molecule; EGF, epidermal growth factor; PDGF, platelet-derived growth factor; ANP, atrial natriuretic peptide; ST, heat-stable enterotoxin of E. coli.For further explanations see text. TABLE

1. Structures

and regulators

of guanylyl

cyclases

Localization

distinguished

by chemical

A membrane-bound form of guanylyl purified from sea urchin sperm (17), cells tivity of particulate guanylyl cyclase that soluble form of the enzyme. In contrast zyme, membrane-bound guanylyl cyclase sist of one subunit, which is glycosylated

withknown primary

GC-B

GC-C

BNP>ANP Mammalian tissues

ST Mammalian intestine

Structure

Monomer

Monomer

Monomer

Monomer

NO Mammalian Bovine

Properties Transmembrane domains Cyclase domains 4ANP,

atrial

115 Glycoprotein 1

1

1

natriuretic

peptide; BNP, brain natriuretic

lung

Dimer

70

120-180

123 Glycoprotein 1

cyclase was first with very high acdo not contain a to the soluble enwas shown to conand exhibits an Mr

Soluble

ANP>BNP Mammalian tissues

135

receptor

structures4

GC-A

gel

and

with were

Purification

Resact Sea urchin

[kDaDNAded

cross-linking

Membrane-bound

Subform

and interact receptors that

purification (9). The less abundant receptor subtype was shown to activate the membrane-bound guanylyl cyclase selectively as the increase in cGMP-forming activity was restricted to membrane preparations (10). The hormoneinduced stimulation of cGMP formation can be markedly potentiated by the addition of ATP or its analog, adenosine-5’-O-(3’-thiotriphosphate) (11). Heat-stable enterotoxins are small peptides produced by Esc/zerichia coli, and cause diarrhea. These peptides stimulate a membranebound guanylyl cyclase in the intestinal mucosa, resulting in marked elevations of cGMP and subsequent effects on chloride and other ion transport (12). No physiological activator of the enzyme has been identified as yet. The occurrence of the heat-stable enterotoxin-activated guanylyl cyclase may not be limited to intestinal mucosa, as in the kidneys of opossum and kangaroo, and in certain colon carcinoma cells the heat-stable enterotoxin also leads to elevation of cyclic GMP levels (13). Calcium appears to be a possible regulator of the membrane-bound guanylyl cyclases occurring in rod outer segments (14) and in unicellular organisms such as Paramecia and Tetrahymena (15). Visual excitation in retinal rod cells leads to increased hydrolysis of cyclic GMP and the consequent closure of cyclic GMP-activated cation-channels, whereby the influx of Na and Ca25. is blocked (16). The lowering of cytosolic Ca2 stimulates the retinal guanylyl cyclase, and through resynthesis of cyclic GMP the dark state is recovered. The inhibitory effect of Ca25. on guanylyl cyclase activity is mediated by a Ca2-sensitive regulatory protein (14). In contrast to the effect on the enzyme in retina, Ca2 leads to stimulation of membrane-bound guanylyl cyclase in protozoans; the Ca2-sensitive protein mediating the response to the cation appears to be calmodulin (15); Ca2Vcalmodulinactivated guanylyl cyclases have so far not been detected in vertebrates.

Activators Source

[M,_.SDS

natriuresis, peptide

114 Glycoprotein ? 1

121 Glycoprotein? 1

1

1

peptide; ST, heat-stable enterotoxin

tissues

Rat lung

Rat kidney

Dimer

73

+

70 + 77 Hemoprotein

70

+

82

70

+

77

76

+

?

-

1+ 1

?

-

1

+

1

of E. co/i.

2786 Vol. 5 October 1991 KOESLING ET AL. The FASEB Journal om www.fasebj.org by Tulane Univ Howard-Tilton Lib (129.81.226.78) on December 02, 2018. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNumber()},

of about 135 kDa on sodium dodecyl sulfate (SDS) gels. Isolation of the membrane-bound guanylyl cyclase from mammalian tissues yielded a glycosylated enzyme with an M of 120-180 kDa and resulted in copurification with an ANP receptor subtype. Whereas the enzyme was shown to bind ANF, this binding was not associated with increased enzymatic activity of the purified enzyme (18, 19). Attempts to purify the enterotoxin-activated guanylyl cyclase and the Ca2-regulated guanylyl cyclases have failed so far. Primary

structure

of membrane-bound

guanylyl

cyclase

Three mammalian membrane-bound guanylyl cyclases (GC-A, GC-B, GC-C) and a sea urchin enzyme have been identified and sequenced (20-25). The amino acid sequences predict proteins with one transmembrane domain that are structurally related to the protein-tyrosine kinase-linked growth factor receptors, with an NH2-terminal extracellular peptide-binding domain, one transmembrane domain, and a COOH-terminal cytosolic region. Besides the structural similarities, the membrane-bound guanylyl cyclases show weak homology to the cytosolic region of the growth factor receptors, which is supposed to be responsible for the tyrosine kinase activity (26). To compare the sequences of the membrane-bound guanylyl cyclases, the polypeptide chains are divided into three domains: the NH2-terminal ligand-binding domain, the intracellular kinase-like domain, and the putative catalytic domain, which is also found in the subunits of soluble guanylyl cyclase and in two hydrophilic domains of the adenylyl cyclases (27) (Fig. 2). The guanylyl cyclase domains reveal the highest degree of similarities with 91% identical amino acids shared by GC-A and GC-B and 55% with GCC (see Fig. 2). Seventy-two percent of identical amino acids are found in the protein tyrosine kinase-like domains of GC-A and GC-B, whereas GC-C reveals 39 and 35% of identities compared with GC-A and GC-B, respectively. The homologies in the extracellular ligand-binding domain are clearly lower, 43% between GC-A and GC-B and 10% between these forms and GC-C. The membrane-bound guanylyl cyclase of the sea urchin shows homologies to the mammalian enzymes in the intracellular part of the molecules whereas the extracellular ligand-binding domain appears to be unrelated. These numbers demonstrate the close relationship between GC-A and GC-B, and suggest that GC-C has branched off earlier during evolution and developed separately. As already suggested by differences in the extracellular ligand-binding domain, the membrane-bound guanylyl cyclases were shown to be activated by different hormones in expression experiments. GC-A serves as a receptor for ANP, and cyclic GMPforming activity was increased by hormone binding (20, 21). The enterotoxin of E. coliwas shown to bind to GC-C and to increase catalytic activity (24). Although GC-B was stimulated by natriuretic peptides, showing a higher affinity for BNP than for ANP, the relatively high concentrations required for activation make it uncertain that either one of the peptides represents the natural ligand (22, 23).

SOLUBLE

GUANYLYL

CYCLASE

Regulation Despite the ability of various hormones and neurotransmitters to elevate cGMP in intact cells, no agent was known to reproducibly stimulate guanylyl cyclase activity in broken

GUANYLYL

CYCLASES

cell preparations, nitric oxide, and

until the late 1970s when sodium azide, sodium nitroprusside were shown to be potent activators of the soluble enzyme (28, 29). As shown in 1981 by Gerzer et al. (30), the soluble enzyme purified from bovine lung contained heme as a prosthetic group, and the interaction of NO-containing compounds or liberated NO with the enzyme-associated heme group was proposed as a model for activation of the enzyme. Nevertheless, the physiological significance of NO-induced activation of soluble guanylyl cyclase was not clear until the endothelium-derived relaxing factor (EDRF), which causes vasodilation via stimulation of soluble guanylyl cyclase and subsequent elevation of cyclic GMP concentrations, was identified as NO (31). Originally, EDRF biosynthesis was shown in endotheliai cells under the influence of vasodilatory factors such as acetylcholine, histamin, and bradykinin (32); in the meantime, the formation of NO from arginine has been demonstrated in a variety of tissues (33). A NO-forming enzyme, designated NO synthase, was purified from cerebellum and shown to depend on Ca2/calmodulin and NADPH (34, 35). Recently, this enzyme was shown to catalyze the conversion of L-arginine into NO and citrulline with tetrahydrobiopterine as a cofactor (36). Therefore, intracellular Ca25. appears to be the hormone-induced signal to stimulate NO synthase, leading to an increase in NO synthesis and stimulation of soluble guanylyl cyclase. Purification Soluble guanylyl cyclase was purified to apparent homogeneity in several laboratories and shown to consist of two different subunits. In recent years, two groups developed immunoaffinity purification procedures and obtained enzymes with 70- and 82-kDa subunits from rat lung (37) and 70- and 73-kDa subunits from bovine lung (38); it was not clear whether the larger subunits obtained by both purification procedures were identical. Meanwhile, sequence analysis of the 73- and 82-kDa subunit (27, 39) revealed that both subunits share 84.6% identical amino acids, consist of 691 amino acids, and have a calculated molecular mass of 77.5, and therefore appear to represent the same subunit.

Primary

structure

Soluble guanylyl cyclase has been purified as a heterodimer, and so far three subunits have been sequenced; two of them, a1 (27, 39) and i3, (40, 41), correspond to the larger and smaller subunits, respectively, of soluble guanylyl cyclase purified from lung, as their sequences were determined with the help of partial peptide sequences obtained from the isolated enzyme. The third subunit (42) was detected using the conservation of amino acids in the putative catalytic domain of the guanylyl cyclases in the polymerase chain reaction. This sequence revealed more similarity toward the i3- than the a1-subunit, and was therefore called /3). The /32-subunit is preferentially expressed in kidney and liver, whereas the mRNA for both the a1- and /31-subunits are most abundant in lung and brain. A comparison of the amino acid sequences of the bovine j3- (70 kDa) and a1- (73 kDa) subunits revealed strong similarities between the subunits with about 32% identical amino acids over the whole sequences (27). The NH2terminal region (about 300 amino acids) shows rather low similarity with 20% identical amino acids; in the following 54 amino acids, the homology among these subunits of soluble guanylyl cyclase is most pronounced (70% identical amino acids). The similarity continues with about 40% of

2787

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Guanylyl cyclases, a growing family of signal-transducing enzymes.

Guanylyl cyclases, which catalyze the formation of the intracellular signal molecule cyclic GMP from GTP, display structural features similar to other...
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