Guanylate Cyclases

Cyclic GMP is synthesized from GTP by the enzyme guanylate cyclase (EC 4.6.1.2) and yields pyrophosphate as a product. Cyclic GMP (cGMP) acts as a second messenger, activating cGMP-dependent protein kinases (see Phosphorylation protein) and/or regulating cGMP-sensitive gated ion channels. The role of cGMP as an intracellular messenger in vascular smooth muscle relaxation and retinal photo-transduction is well established (see -Monophosphate, cGMP)). Garbers, Goeddel and co-workers (1-3) found that the catalytic centers of guanylyl cyclases are strongly related to eukaryotic class III adenylate cyclases. Thus guanylate cyclases form a single class, in contrast to the four classes of adenylate cyclases, but they are of at least two very different types, linked to the function of the enzyme in the cell and either soluble or membrane-bound in eukaryotic cells. These two forms differ in their structure, regulation, biochemical, and physicochemical properties (4-7. (

1. Membrane-bound Guanylate Cyclases

 Guanylate cyclases in family I act as sensors and are often receptors for hormones, such as atrial natriuretic peptide (ANP), which is involved in controlling of osmotic pressure and sodium excretion in mammals. These guanylate cyclases are complex, membrane-bound enzymes comprising a receptor for specific hormones coupled to a catalytic domain similar in sequence and structure to the catalytic domain of class III adenylate cyclases (1-3). The known guanylate cyclase receptors form several subfamilies (2, 6,8). Those from sea urchins recognize speract and resact , small peptides that stimulate sperm motility and metabolism ((9),(10)). The receptors for natriuretic peptides (ANF( exist in two forms, both of which synthesize cGMP: guanylate cyclase-A (also named ANP-A), which is specific for ANP, and guanylate cyclase-B (or ANP-B) which is stimulated by brain natriuretic peptide rather than by ANP. There are at least three ANP receptors, two with guanylate cyclase activity (ANP-A and ANP-B) and one (ANP-C) that is responsible for clearing ANP from the circulation without a role in signal transduction (11). Intestinal cells contain the receptor for the Escherichia coli heat-stable enterotoxin (guanylate cyclase C). The endogenous ligand for this intestinal receptor is a small peptide called guanylin .

Odorant information is encoded by a series of intracellular signal transduction events thought to be mediated primarily by the second messenger cAMP. But a subset of olfactory neurons expresses a cGMP-stimulated phosphodiesterase (PDE2) and a guanylate cyclase of the receptor type (guanylate cyclase D), which demonstrate that cGMP has an important regulatory function in olfactory signaling (see -Monophosphate, cGMP)) (8). Finally, retinal guanylate cyclases (often named retGC) exist in at least two forms, retGC-E and retGC-F. They play a specific functional role in the rods and/or cones of photoreceptors and trigger a protein phosphorylation cascade (12). They consist of an apparent extracellular domain linked by a single transmembrane region to an intracellular domain. They are coupled to guanylate cyclase activating protein-2, which is a  -binding protein that activates retGC-1 in a  -sensitive manner. It is not known whether retGCs act as receptors, but their structures are similar to that of the other plasma membrane-bound guanylate cyclases.

The organization of all these guanylate cyclases is similar: they have an N-terminal extracellular domain that acts as the ligand-binding receptor region, then a transmembrane region, followed by a large cytoplasmic C-terminal region that can be subdivided into two domains, a guanylate cyclase catalytic domain and a protein kinase-like domain that is important for controlling the protein phosphorylation cascade linked to the specific signal recognized by the cognate guanylate cyclase (13, 14) .

2. Soluble guanylate cyclases

The second family of guanylate cyclases is cytoplasmic and soluble and often designated sGC. They have completely different regulatory functions and always form heterodimers. The two subunits, alpha and beta, are proteins that, although different in length (from 70 to 82 kDa) and sequence, are highly related (15). Two forms of beta subunits are currently known, beta-1, which is expressed in lung and brain, and beta-2, which is more abundant in kidney and liver. The most fascinating feature of these subunits is that they bind a heme prosthetic group. Upon binding of nitric oxide (NO) to this heme group, the sGC catalytic activity is stimulated and generates the intracellular signaling molecule cGMP. NO, discovered in 1987, is a signal transduction molecule. Its importance has been emphasized by its role in blood circulation and cardiac muscle functioning (16, 17). Carbon monoxide (CO) also plays a role similar to that of NO (18).

Fifteen conserved cysteine residues of sGC have been mutated to serine by in vitro site-directed mutagenesis . All of the resulting recombinant enzymes synthesize cGMP, demonstrating that these residues are not directly involved in catalysis. On the other hand, mutation of two cysteine residues located in the N-terminal, putative heme-binding region of the beta subunit yields proteins that are insensitive to NO and lose their heme prosthetic group. In contrast, mutation of the corresponding cysteine residues of the alpha subunit does not alter their NO responsiveness, indicating that heme binding is probably a specific feature of the N-terminal domain of the beta subunit (19. (

3. Similarities Between the Membrane and Soluble Guanylate Cyclases and Class III Adenylate Cyclases

 In general, the genetic organizations of both enzyme types have been conserved by the localization of at least one catalytic domain in the carboxy-terminal part of the protein, coupled to a variety (in length and in sequence) of amino-terminal parts. Both forms of guanylate cyclases share a conserved domain that is fundamental for the catalytic activity of the enzyme. A similar domain is also found twice in the different forms of membrane-bound, class III adenylate cyclases from mammals, slime mold, or Drosophila. A polypeptide consensus pattern detects both domains of class-III adenylate kinases and guanylate cyclases: Gly-Val-[Leu/Ile/Val/Met]-X_0,1-Gly-X5-[Phe/Tyr]-X-[Leu/Ile/Val/Met]-[Phe/Tyr/Trp]-[Gly/Ser]-[Asp/Asn/Thr/His/Lys/Trp]-[Asp/Asn/Thr]-[Ile/Val]-[Asp/Asn/Thr/Ala]-X₅-[Asp/Glu] (1, 3. (

The common evolutionary origin of the adenylate cyclases and guanylate cyclases, evidenced by the apparent facility with which it is possible to build up a purine nucleoside triphosphate cyclase of broad specificity, might be relevant to the phylogeny of the catalytic center of class III adenylate cyclases (20). This similarity suggests the existence of a common ancestral purine nucleotide triphosphate cyclase. As a consequence of this interpretation of the sequence data, one might wonder whether evolution has not permitted the existence of some overlap in their specificities because a given cyclase is triggered, under appropriate regulatory conditions, to synthesize either cAMP or cGMP alternatively. This could have been used in cyclic nucleotide-mediated controls existing in eukaryotes, and it might explain the older observations that, in some cases at least, the cAMP and cGMP concentrations vary in opposite directions.

References

1. M. S. Chang, D. G. Lowe, M. Lewis, R. Hellmiss, E. Chen, and D. V. Goeddel, (1989) Nature .341. 68-71.

2. D. L. Garbers, (1992) Cell 71, 1–4. 

3. O. Barzu and A. Danchin (1994) Prog. Nucleic Acids Res. Mol. Biol. 49, 241–283. 

4. D. L. Garbers (1990) The New Biologist, 2, 499–504. 

5. D. Koesling, E. Böhme, and G. Schultz, (1991) FASEB J. 5, 2785–2791. 

6. S. Schulz, M. Chinkers, and D. L. Garbers, (1989) FASEB J. 3, 2026–2035. 

7. S. Schulz, P. S. T. Yuen, and D. L. Garbers, (1991) Trends Pharm. Sci. 12, 116–120. 

8. S. Yu, L. Avery, E. Baude, and D. L. Garbers, (1997) Proc. Natl Acad. Sci. USA 94, 3384–3387.

9. D. L. Garbers, J. K. Bentley, L. J. Dangott, C. S. Ramarao, H. Shimomura, N. Suzuki, and D.ferences Thorpe, (1986) Adv. Exp. Med. Biol. 207, 315–357. 

10. H. Shimomura, L. J. Dangott, and D. L. Garbers, (1986) J. Biol. Chem., 261, 15778–15782. 

11. M. Chinkers and D. L. Garbers (1991) Adv. Cyclic Nucleic Acid Res. 60, 553–575. 

12. R. B. Yang, D. C. Foster, D. L. Garbers, and H. J. Fulle (1995) Proc. Natl. Acad. Sci. USA 92, 602-606.

13. J. G. Aparicio and M. L. Applebury (1996) J. Biol. Chem. 271, 27083–27089. 

14. D. Koesling, G. Schultz, and E. Böhme (1991) FEBS Lett. 280, 301–306. 

15.B. Wedel, C. Harteneck, J. Foerster, A. Friebe, G. Schultz, and D. Koesling, (1995) J. Biol. Chem. 270, 24871–24875. 

16. H. Kook, S. E. Lee, Y. H. Baik, S. S. Chung, and J. H. Rhee, (1996) Life Sciences 59, 41–47. 

17. O. W. Griffith and R. G. Kilbourn (1997) Adv. Enzyme Regul. 37, 171–194. 

18. A. Friebe, G. Schultz, and D. Koesling, (1996) EMBO J. 15, 6863–6868. 

19. A. Friebe, B. Wedel, C. Harteneck, J. Foerster, G. Schultz, and D. Koesling, (1997( Biochemistry 36, 1194–1198. 

20. A. Beuve and A. Danchin (1992) J. Mol. Biol. 225, 933–938.