Functional consequences of a rod outer segment membrane guanylate cyclase (ROS-GC1) gene mutation linked with Leber's congenital amaurosis.

ROS-GC1 is the original member of the subfamily of membrane guanylate cyclases with two Ca2+ switches, which have been defined as CRM1 and CRM2. These are separately located within the intracellular domain of the cyclase. CRM1 switches on the enzyme at nanomolar concentrations of Ca2+ and is linked with phototransduction; the other stimulates at micromolar Ca2+ concentrations and is predicted to be linked with retinal synaptic activity. Ca2+ acts indirectly via Ca2+-binding proteins, GCAP1 and CD-GCAP. GCAP1 is a modulator of the CRM1 switch, and CD-GCAP turns on the CRM2 switch. A Leber's congenital amaurosis, termed LCA1, involves F514S point mutation in ROS-GC1. The present study shows that the mutation severely damages its intrinsic cyclase activity and inactivates its CRM1 switch but does not affect the CRM2 switch. In addition, on the basis of the established modulatory features of ROS-GC1, it is predicted that, in two other forms of LCA1 involving deletion of nt 460C or 693C, there is a frameshift in ROS-GC1 gene, which results in the nonexpression of the cyclase. For the first time, the findings define the linkage of distinct molecular forms of LCA to ROS-GC1 in precise biochemical terms; they also explain the reasons for the insufficient production of cyclic GMP in photoreceptors to sustain phototransduction, which ultimately leads to the degeneration of the photoreceptors.

Calcium modulated signaling site in type 2 rod outer segment membrane guanylate cyclase (ROS-GC2).

The ROS-GC subfamily of membrane guanylate cyclases is at present represented by two members: ROS-GC1 and ROS-GC2. A unique functional feature of this subfamily is that it is intracellularly modulated in low Ca2+ concentration by calmodulin-like Ca(2+)-binding proteins termed GCAPs, 1 and 2, and the modulation is consistent with its linkage to phototransduction. The present study shows that: (1) GCAP2 is a specific modulator of ROS-GC2; (2) through systematic remodeling of ROS-GC modules, the study also shows that the modulated domain resides within the amino acid segment 736-1020. This domain is distinct form the corresponding GCAP1-modulated ROS-GC1 domain. Thus, GCAP1 and GCAP2 act through different ROS-GCs and through two different cyclase domains.

Third calcium-modulated rod outer segment membrane guanylate cyclase transduction mechanism.

Ca2+-modulated rod outer segment membrane guanylate cyclase (ROS-GC1) has been cloned and reconstituted to show that it is regulated by two processes: one inhibitory, the other stimulatory. The inhibitory process is consistent with its linkage to phototransduction; the physiology of the stimulatory process is probably linked to neuronal transmission. In both regulatory processes, calcium modulation of the cyclase takes place through the calcium binding proteins; guanylate cyclase activating proteins (GCAP1 and GCAP2) in the case of the phototransduction process and calcium-dependent GCAP (CD-GCAP) in the case of the stimulatory process. The cyclase domains involved in the two processes are located at two different sites on the ROS-GC1 intracellular region. The GCAP1-modulated domain resides within the aa 447-730 segment of ROS-GC1 and the CD-GCAP-modulated domain resides within the aa 731-1054 segment. In the present study the GCAP2-dependent Ca2+ modulation of the cyclase activity has been reconstituted using recombinant forms of GCAP2 and ROS-GC1, and its mutants. The results indicate that consistent to phototransduction, GCAP2 at low Ca2+ concentration (10 nM) maximally stimulates the cyclase activity of the wild-type and its mutants: ext (deleted aa 8-408), kin (deleted aa 447-730) and hybrid consisting of the ext, transmembrane and kin domains of ANF-RGC and the C-terminal domain, aa 731-1054, of ROS-GC1. In all cases, it inhibits the cyclase activity with an IC50 of about 140 nM. A previous study has shown that under identical conditions the kin and the hybrid mutant are at best only minimally stimulated. Thus, the GCAP1 and GCAP2 signal transduction mechanisms are different, occurring through different modules of ROS-GC1. These findings also demonstrate that the intracellular region of ROS-GC1 is composed of multiple modules, each designed to mediate a particular calcium-specific signalling pathway.

Differential activation of rod outer segment membrane guanylate cyclases, ROS-GC1 and ROS-GC2, by CD-GCAP and identification of the signaling domain.

The ROS-GC is one of the two subfamilies of membrane guanylate cyclases. It distinguishes itself from the other surface receptor subfamily in that its members are not regulated by extracellular peptides; instead, they are modulated by intracellular Ca2+ signals. There are two members of the subfamily, ROS-GC1 and ROS-GC2. An intriguing feature of ROS-GC1 is that it has two Ca2+ switches. One switch inhibits the enzyme at micromolar concentrations of Ca2+, and the other stimulates. The inhibitory switch is linked to phototransduction, and it is likely that the stimulatory switch is linked to retinal synaptic activity. Ca2+ acts indirectly via Ca(2+)-binding proteins, GCAPs and CD-GCAP. GCAPs modulate the inhibitory switching component of the cyclase and CD-GCAP turns on the activation signaling switch. The activating switch of ROS-GC2 has not so far been scrutinized. The present study shows that CD-GCAP is linked to the activation signaling switch of ROS-GC2, but the linkage is about 10-fold weaker than that of the ROS-GC1. Thus, CD-GCAP is a specific ROS-GC1 activator. Furthermore, through a series of expression studies on the mutants involving deletion, building of hybrids, and reconstruction of a heterologous cyclase, the study confirms that the CD-GCAP regulated switch resides within the amino acid segment 736-1053 of the cyclase.

Structural and functional characterization of retinal calcium-dependent guanylate cyclase activator protein (CD-GCAP): identity with S100beta protein.

Calcium-dependent guanylate cyclase activator protein (CD-GCAP) is a low-molecular-weight retinal calcium-binding protein which activates rod outer segment guanylate cyclase (ROS-GC) in a calcium-dependent manner. This investigation was undertaken to determine the protein's structure and identity. Partial amino acid sequencing (72% of the protein), mass spectral analysis, cloning, and immunological studies revealed that CD-GCAP is identical to S100beta, another low-molecular-weight calcium-binding protein whose structure was known. We had shown earlier that the latter protein, which is usually called S100b (S100betabeta or dimer of S100beta), also activates ROS-GC but that the Vmax of activated cyclase was about 50% lower than when stimulated by CD-GCAP. S100b also required about 15 times more calcium (3.2 x 10(-)5 vs 1.5 x 10(-)6 M) for half-maximal stimulation of cyclase. To investigate the possibility that CD-GCAP is a post-translationally modified form of S100b, both proteins were treated with 1 M hydroxylamine which is known to deacylate proteins. After the treatment, CD-GCAP did not activate cyclase while S100b activation remained unaffected suggesting that CD-GCAP could not be a modified form of S100b. Hydroxylamine also broke down CD-GCAP into smaller fragments while leaving S100b intact. It therefore appeared that in spite of identical primary structures, the conformations of the two proteins were different. We then investigated the possibility that the purification procedures of the two proteins, which were quite different, could have contributed to such conformational differences: CD-GCAP purification included a step of heating at 75 degrees C in 5 mM Ca, while S100b purification included zinc affinity chromatography. To test the influence of these treatments on the properties of the proteins, CD-GCAP was subjected to zinc affinity chromatography and purified as S100b (CD-GCAP-->S100b) and S100b was heated in Ca and purified as CD-GCAP (S100b-->CD-GCAP). Cyclase activation, calcium-sensitivity, and hydroxylamine-lability measurements revealed that CD-GCAP-->S100b is identical to S100b and that S100b-->CD-GCAP is identical to CD-GCAP. Taken together the results demonstrate that CD-GCAP and S100b are one and the same protein and that their functional differences are due to different interconvertible conformational states.

Structural and functional characterization of a second subfamily member of the calcium-modulated bovine rod outer segment membrane guanylate cyclase, ROS-GC2.

A native bovine calcium-modulated rod outer segment membrane guanylate cyclase (ROS-GC) has been cloned and reconstituted to show its linkage consistent to the process of phototransduction. In the present study, a second form of the membrane guanylate cyclase has been cloned from the bovine retina. This cyclase shares a high sequence identity with ROS-GC, is specifically expressed in the bovine retina, and, like ROS-GC, is modulated in low Ca2+ by a calmodulin-like Ca2+-binding protein, termed GCAP2. For this reason, this cyclase has now been named ROS-GC2 and the previously described ROS-GC as ROS-GC1. The tail end of ROS-GC2 contains a stretch of five amino acids, a structural feature unique to itself. These findings support the existence of a calcium-modulated subfamily of ROS-GC and indicate that ROS-GC2 embodies a five amino acid signature element at its tail end.

Membrane guanylate cyclase signal transduction system.

The suspect role of the receptor-mediated cyclic GMP signaling pathway was dispelled by the discovery of a membrane guanylate cyclase that was also an atrial natriuretic factor receptor. It is now established that the membrane guanylate cyclase transduction system is linked to the signaling of natriuretic factors, guanylin/enterotoxin, and emerging evidence suggests that a new neural tissue-specific subfamily of membrane guanylate cyclases exists whose mechanism of signal transduction is different from those of the other membrane cyclases. This review will briefly discuss the fascinating, albeit turbulent, history of this signal transduction field, which will be followed by its current status and finally the direction it is heading.

Distinct inhibitory ATP-regulated modulatory domain (ARMi) in membrane guanylate cyclases.

Depending upon the cofactors Mg2+ or Mn2+, ATP stimulates or inhibits the signal transduction activities of the natriuretic factor receptor guanylate cyclases, ANF-RGC and CNP-RGC: there is stimulation in the presence of Mg2+ and inhibition in the presence of Mn2+. A defined core ATP-regulated modulatory (ARM) sequence motif within the intracellular 'kinase-like' domain of the cyclases is critical for stimulation, but the mechanism of the inhibitory transduction process is not known. In addition, ATP inhibits the basal cyclase activity of a rod outer segment membrane guanylate cyclase (ROS-GC). The mechanism of this inhibitory transduction process is also not known. These issues have been addressed in the present investigation through a program of deletion mutagenesis/expression studies of the cyclases. The study shows that the ATP-mediated inhibitory transduction processes of the natriuretic factor receptor cyclases and of ROS-GC are identical. The ATP-regulated inhibitory domain of all these cyclases resides within the C-terminal segment of the cyclase. This domain is in a different location from the one representing the ATP-stimulatory ARM. The identification of the inhibitory domain in the C-terminal segment of the cyclase indicates that this segment is composed of two separate domains: one representing a catalytic cyclase domain and the other an ATP-regulated inhibitory (ARMi) domain. These findings establish a novel ATP-mediated inhibitory transduction mechanism of the membrane guanylate cyclases which is distinct from that of its counterpart, the stimulatory ATP-mediated hormonal signal transduction mechanism. Thus, they define a new paradigm of guanylate cyclase-linked signal transduction pathways.

Calcium modulation of bovine photoreceptor guanylate cyclase.

Bovine photoreceptor guanylate cyclase (ROS-GC) consists of a single transmembrane polypeptide chain with extracellular and intracellular domains. In contrast to non-photoreceptor guanylate cyclases (GCs) which are activated by hormone peptides, ROS-GC is modulated in low Ca2+ by calmodulin-like Ca(2+)-binding proteins termed GCAPs (guanylate cyclase-activating proteins). In this communication we show that, like the native system, ROS-GC expressed in COS cells is activated 4-6-fold by recombinant GCAP1 at 10 nM Ca2+ and that the reconstituted system is inhibited at physiological levels of Ca2+ (1 microM). A mutant ROS-GC in which the extracellular domain was deleted was stimulated by GCAP1 indistinguishable from native ROS-GC indicating that this domain is not involved in Ca2+ modulation. Deletion of the intracellular kinase-like domain diminished the stimulation by GCAP1, indicating that this domain is at least in part involved in Ca2+ modulation. Replacement of the catalytic domain in a non-photoreceptor GC by the catalytic domain of ROS-GC yielded a chimeric GC that was sensitive to ANF/ATP and to a lesser extent to GCAP1. The results establish that GCAP1 acts at an intracellular domain, suggesting a mechanism of photoreceptor GC stimulation fundamentally distinct from hormone peptide stimulation of other cyclase receptors.

Molecular characterization of S100A1-S100B protein in retina and its activation mechanism of bovine photoreceptor guanylate cyclase.

In contrast to the membrane guanylate cyclases which are stimulated by extracellular ligands, rod outer segment membrane guanylate cyclase (ROS-GC) activity is modulated intracellularly by calcium in two ways: one, where it is inhibited, and the other, where it is stimulated. The former way is linked to the phototransduction, and physiology of the second is unknown. In both ways calcium modulation of the cyclase occurs through the calcium binding proteins: through guanylate cyclase activating proteins (GCAPs) in the case of phototransduction, and through the recently discovered calcium-dependent GCAP (CD-GCAP) in the case of the other way. The kinase-like domain of ROS-GC is critical for the phototransduction-linked process. The present study shows the expression of alpha and beta chains of S100A1-S100B protein in the bovine retina and demonstrates that this protein stimulates ROS-GC activity in a dose-dependent fashion, that the stimulation is calcium dependent with an EC50 of 17 microM, and that the kinase-like domain is not involved in the calcium-modulated cyclase activation. Instead the involved domain resides at the C-terminal segment, between amino acids 731 and 1054. Thus, this S100A1-S100B protein-mediated calcium-modulated signal transduction mechanism is novel. Furthermore, this study provides the molecular understanding of the two transduction processes mediated by the same ROS-GC, one linked to the low and the other to the high calcium levels.

Single amino acid residue-linked signaling shifts in the transduction activities of atrial and type C natriuretic factor receptor guanylate cyclases.

The type A (ANF) and the type C (CNP) natriuretic factor-activated guanylate cyclases, respectively termed as ANF-RGC and CNP-RGC, are single-chain transmembrane-spanning proteins, containing ligand binding and catalytic cyclase domains at two opposite ends of the protein. The binding activity resides at the N-terminal extracellular region and the catalytic cyclase activity at the carboxyl end. The ANF-RGC residue Leu-364, residing in the extracellular region, is critical for the ANF-binding activity; the CNP-RGC residue Glu-332 is critical for the CNP-binding activity. The counter part of CNP-RGC-Glu-332 residue is the ANF-RGC residue Gln-338 and of ANF-RGC-Leu-364 residue in CNP-RGC is the Valine-358. The present study shows a remarkable signal switching phenomenon associated with these residues. By changing the ANF-RGC residue Gln-338 to Glu, ANF-RGC switches from no to significant CNP signal transduction activity; similarly, a change from Valine-358 to Leu generates ANF signal transduction activity in CNP-RGC. These acquired signal transduction activities in the cyclases are in addition to their natural signal transduction activities. Thus, these new cyclases show both ANF and CNP signaling activities.

Structural and functional characterization of the rod outer segment membrane guanylate cyclase.

In the vertebrate photoreceptor cell, rod outer segment (ROS) is the site of visual signal-transduction process, and a pivotal molecule that regulates this process is cyclic GMP. Cyclic GMP controls the cationic conductance into the ROS, and light causes a decrease in the conductance by activating hydrolysis of the cyclic nucleotide. The identity of the granylate cyclase (ROS-GC) that synthesizes this pool of cyclic GMP is unknown. We now report the cloning, expression and functional characterization of a DNA from bovine retina that encodes ROS-GC.

Glutamic acid-332 residue of the type C natriuretic peptide receptor guanylate cyclase is important for signaling.

The type C natriuretic peptide (CNP)-activated guanylate cyclase (CNP-RGC) is a single-chain transmembrane-spanning protein, predicted to contain both ligand binding and catalytic activities. Upon binding CNP, CNP-RGC catalyzes the formation of cyclic GMP. We now show that the Glu-332 residue residing in the extracellular region of CNP-RGC plays an important role in signal transduction. Deletion of the CNP-RGC intracellular region resulted in the CNP receptor which lacked cyclase activity; deletion or substitution of Glu-332 with His or Lys resulted in almost total loss of both CNP binding and the CNP-dependent cyclase activity without affecting the basal cyclase activity of the mutant proteins. These observations support the general signal transduction model of the subfamily of natriuretic factor receptor cyclases where it is predicted that ligand binding to the extracellular receptor domain of the protein activates the cytosolic catalytic domain, generating the second-messenger cyclic GMP, and identify an amino acid residue of CNP-RGC that plays an important role in CNP signaling.

The glycine residue of ATP regulatory module in receptor guanylate cyclases that is essential in natriuretic factor signaling.

Atrial natriuretic factor (ANF) and C-type natriuretic peptide (CNP)-activated guanylate cyclases are single-chain transmembrane-spanning proteins, containing both ligand binding and catalytic activities. In both proteins, ligand binding to the extracellular receptor domain activates the cytosolic catalytic domain, generating the second messenger cyclic GMP. Obligatory in this activation process is an ATP-dependent step. ATP directly binds to a defined ATP-regulatory module (ARM) sequence motif in the cyclases and through ARM bridges the events of ligand binding and signal transduction. These ARM sequence motifs are respectively represented by Gly503-Xa-Gly505-Xa-Xa-Xa-Gly509 and Gly499-Xa-Xa-Xa-Gly503 in the case of ANF receptor guanylate cyclase (ANF-RGC) and CNP receptor guanylate cyclase (CNP-RGC). Through genetic remodeling techniques, we now show that ARM-Gly505 in ANF-RGC and the corresponding ARM-Gly499 in CNP-RGC are critical for ANF and CNP signaling, and other ARM-Gly residues have minimal effect in the respective signaling processes.

Structural and biochemical identity of retinal rod outer segment membrane guanylate cyclase.

Recent molecular cloning reports show that there are at least three membrane guanylate cyclases in vertebrate retina: (1) atrial natriuretic factor receptor guanylate cyclase (ANF-RGC), (2) C-type natriuretic peptide receptor guanylate cyclase (CNP-RGC), and (3) "retinal guanylate cyclase" (RetGC). The specific cellular localization of the first two cyclases is unknown, but RetGC is apparently localized in photoreceptor cells, suggesting that it participates in visual transduction. With the overall objective of identifying the guanylate cyclase that is linked to phototransduction, we compared the structural and regulatory properties of the biochemically characterized 112 kDa bovine rod outer segment membrane guanylate cyclase (ROS-GC) with those of RetGC, ANF-RGC and CNP-RGC. The N-terminal and two internal peptide sequences of purified ROS-GC had about 90% similarity with the corresponding sequences of the RetGC; the sequence identity with natriuretic peptide receptor cyclases was about 30%. A 19 amino acid long sequence from a tryptic peptide of ROS-GC had no corresponding sequence in the other three cyclases. ROS-GC was inhibited by ATP but ANF-RGC and CNP-RGC were activated by ATP in the presence of the respective peptide hormones. These results suggest that ROS-GC represents a new subtype of the membrane guanylate cyclase family that is structurally and biochemically distinct from the other retinal cyclases.

Cloning and expression of an ATP-regulated human retina C-type natriuretic factor receptor guanylate cyclase.

The natriuretic factors are structurally related polypeptide hormones that regulate the hemodynamics of the physiological processes of diuresis, water balance, and blood pressure. Presumably, these hormones act through the activation of guanylate cyclases which are also the specific receptors of these hormones. Two such structurally similar cell surface receptors are known; the ligand for one is atrial natriuretic factor (ANF) and for the other is C-type natriuretic peptide (CNP). Studies with ANF receptor guanylate cyclase (ANF-RGC) have indicated that its ligand binding site is extracellular and the catalytic site is intracellular, but the mere ligand binding to the receptor domain does not activate the cytosolic catalytic domain. An intervening ATP-mediated event is obligatory: ATP binds to a defined ATP-regulated module (ARM) sequence and bridges the events of ligand binding and signal transduction. The mechanism of CNP signaling is not known, although CNP in intact cells transfected with CNP receptor guanylate cyclase (CNP-RGC) stimulates the formation of cyclic GMP. Furthermore, there is no prior evidence of the presence of CNP signal transduction system in retina, although the presence of ANF-RGC has been documented. We now report the molecular cloning and expression of CNP-RGC from human retina and show that ATP is obligatory in CNP signaling also.(ABSTRACT TRUNCATED AT 250 WORDS)

Core sequence of ATP regulatory module in receptor guanylate cyclases.

Atrial natriuretic factor (ANF) and C-type natriuretic peptide (CNP)-activated guanylate cyclases are single-chain transmembrane-spanning proteins, containing both ligand binding and catalytic activities. In both proteins, ligand binding to the extracellular receptor domain activates the cytosolic catalytic domain, generating the second messenger cyclic GMP. Studies with ANF receptor guanylate cyclase (ANF-RGC) have indicated that obligatory in this activation process is an ATP-dependent step. ATP directly binds to the cyclase and bridges the events of ligand binding and signal transduction. A defined ATP-regulated module (ARM) sequence (Gly503-Arg-Gly-Ser-Asn-Tyr-Gly509) in the cyclase is critical in the ATP-mediated event. Through genetic remodeling techniques, we have now identified the core ARM sequence that is essential in both ANF and CNP signaling. This sequence is Gly-Xa-Xa-Xa-Gly, represented by Gly505-Ser-Asn-Tyr-Gly509 in the case of ANF-RGC ARM and by Gly499-Ser-Ser-Tyr-Gly503 in the CNP receptor guanylate cyclase ARM.

A structural motif that defines the ATP-regulatory module of guanylate cyclase in atrial natriuretic factor signalling.

Atrial natriuretic factor (ANF)-dependent guanylate cyclase is a single-chain transmembrane-spanning protein, containing an ANF receptor and having catalytic activity. ANF binding to the receptor domain activates the catalytic domain, generating the second messenger cyclic GMP. Obligatory in this activation process is an intervening step regulated by ATP, but its mechanism is not known. Through a programme of site-directed and deletion mutagenesis/expression studies, we report herein the identity of a structural motif (Gly503-Arg-Gly-Ser-Asn-Tyr-Gly509) that binds ATP and amplifies the ANF-dependent cyclase activity; this, therefore, represents an ATP-regulatory module (ARM) of the enzyme, which plays a pivotal role in ANF signalling.

Genetically tailored atrial natriuretic factor-dependent guanylate cyclase. Immunological and functional identity with 180 kDa membrane guanylate cyclase and ATP signaling site.

Biochemical and immunological studies have established that one of the signal transducers of atrial natriuretic factor (ANF) is a 180 kDa membrane guanylate cyclase (180 kDa mGC), which is also an ANF receptor; obligatory in the transduction process is an intervening ATP-regulated step, but its mechanism is not known. GC alpha is a newly discovered member of the guanylate cyclase family whose activity is independent of the known natriuretic peptides, and the enzyme is not an ANF receptor. The genetically tailored GC alpha, GC alpha-DmutGln338Leu364, however, is not only a guanylate cyclase but also an ANF receptor and is structurally and functionally identical to the cloned wild-type ANF receptor guanylate cyclase, GC-A. We now report that the ANF-dependent guanylate cyclase activity in the particulate fractions of cells transfected with GC alpha-DmutGln338Leu364 was inhibited by the 180 kDa mGC polyclonal antibody, and with this antibody probe it was possible to purify the 130 kDa expressed receptor; the hormone-dependent cyclase activity of this receptor was exclusively dependent upon ATP; and through site-directed mutational studies with GC alpha mutants, the signaling sequence that defines ATP binding site was identified. We thus conclude that 180 kDa mGC and the mutant protein are immunologically similar, both proteins are linked to the ANF signal in the generation of cyclic GMP synthesis; and in both the ligand binding and catalytic activities are bridged through a defined ATP binding module.

Site-directed mutational analysis of a membrane guanylate cyclase cDNA reveals the atrial natriuretic factor signaling site.

Natriuretic peptides are structurally related hormones that regulate hemodynamics of the physiological processes of diuresis, water balance, and blood pressure. One of the second messengers of these hormones is cGMP, and the type of receptor that is involved in the generation of cGMP is also a guanylate cyclase. Recent genetic evidence has revealed such a receptor family; two family members, GC-A and GC-B, have been cloned. We now describe the molecular cloning, sequencing, and expression of a cDNA clone from rat adrenal gland that encodes a membrane guanylate cyclase, GC alpha, that, with the exception of two amino acids, is structurally identical to GC-A and conforms to the purported topographical model of GC-A. The two amino acid changes are the substitutions Gln338----His338 and Leu364----Pro364, involving single nucleotide changes, CAG----CAC and CTG----CCG, respectively. Expression studies indicate that GC alpha cyclase activity is independent of the known natriuretic peptides, and direct binding studies demonstrate that GC alpha is not an ANF receptor. To determine the importance of Gln338 and Leu364 in ANF signaling, the GC alpha cDNA regions encoding amino acid residues 338 and 364 were remodeled by oligonucleotide-directed mutagenesis. A double mutant encoding Gln338 and Leu364, and a single-substitution mutant encoding Leu364 expressed both ANF binding and ANF-dependent cyclase activities, but the mutant encoding Gln338 and a deletion mutant lacking residue 364 did not express either of the above activities. These results define the critical role of Leu364 in ANF signal transduction.