A guanylyl cyclase (GC-D) was recently shown to be expressed in a subclass of neurons within the neuroepithelim of the rat, but given that only a single cyclase was discovered, whether it represents an odorant͞pheromone receptor as has been suggested for the large family of seventransmembrane receptors remains unclear. Through cloning and expression of cDNA we now demonstrate that at least 29 genomic or cDNA sequences found in Caenorhabditis elegans represent guanylyl cyclases. Many of the membrane forms retain cysteine residues conserved within the extracellular, ligand-binding domain of known cyclase receptors. Of eight orphan cyclase receptor::GFP (green f luroescence protein) fusion constructs for which signals were obtained, all were expressed in specific sensory neurons. Furthermore, a cyclase͞GFP fusion protein (GCY-10͞GFP) was principally expressed in the sensory cilium, suggesting these cyclases function as primary chemosensory receptors. For the first time, we also found that chemosensory neurons (ASE), known to be bilaterally symmetric, demonstrate absolute right or left sidedness with respect to the expression of three different cyclases. Thus, the guanylyl cyclases represent an unexpectedly large and new family of sensory neuron receptors that may complement the 7-transmembrane family of odorant͞ pheromone receptors.

Two isoforms of the catalytic subunit of cAMPdependent protein kinase, C␣ and C␤1, are known to be widely expressed in mammals. Although much is known about the structure and function of C␣, few studies have addressed the possibility of a distinct role for the C␤ proteins. The present study is a detailed comparison of the biochemical properties of these two isoforms, which were initially expressed in Escherichia coli and purified to homogeneity. C␤1 demonstrated higher K m values for some peptide substrates than did C␣, but C␤1 was insensitive to substrate inhibition, a phenomenon that was observed with C␣ at substrate concentrations above 100 M. C␣ and C␤1 displayed distinct IC 50 values for the ␣ and ␤ isoforms of the protein kinase inhibitor, protein kinase inhibitor (5-24) peptide, and the type II␣ regulatory subunit (RII␣). Of particular interest, purified type II holoenzyme containing C␤1 exhibited a 5-fold lower K a value for cAMP (13 nM) than did type II holoenzyme containing C␣ (63 nM). This latter result was extended to in vivo conditions by employing a transcriptional activation assay. In these experiments, luciferase reporter activity in COS-1 cells expressing RII␣ 2 C␤1 2 holoenzyme was half-maximal at 12-fold lower concentrations of 8-(4-chlorophenylthio)-cAMP and 5-fold lower concentrations of forskolin than in COS-1 cells expressing RII␣ 2 C␣ 2 holoenzyme. These results provide evidence that type II holoenzyme formed with C␤1 is preferentially activated by cAMP in vivo and suggest that activation of the holoenzyme is determined in part by interactions between the regulatory and catalytic subunits that have not been described previously.cAMP exerts its effects in mammalian cells primarily through the activation of cAMP-dependent protein kinase (cAK), 1 a tetrameric enzyme consisting of two catalytic (C) and two regulatory (R) subunits. Occupation of the cyclic nucleotide binding sites of the R subunits by cAMP results in relief of R subunit inhibition of the C subunits and dissociation of the holoenzyme complex. The dissociated, active C subunit can then affect the cell physiology via phosphorylation of a wide variety of protein substrates (1-3).One possible explanation for the diversity of cellular responses to cAMP is the presence of multiple isoforms of cAK. Three C and four R mammalian subunit isoforms have been described to date: C␣, C␤, C␥, RI␣, RI␤, RII␣, and RII␤. Splice variants of the prototypic C␤ protein (C␤1) also have been reported, giving rise to C␤2 and C␤3 isoforms (4, 5). C␣ and C␤1 have been cloned from numerous sources (6 -9), whereas C␥ has only been cloned from human testis and has not been reported in other species (10). The amino acid sequences of C␣ and C␤1 within a given species are 91% identical (7); however, the amino acid identity of C␣ proteins from different species is significantly greater (98 -100%) (7, 11), suggesting that each kinase plays a distinct role(s) in cellular regulation (7,12). Furthermore, C␣ is ubiquitously expressed in mammalian tissues, whereas C␤1...

In mammals, cyclic GMP and cGMP-dependent protein kinases (cGKs) have been implicated in the regulation of many neuronal functions including long-term potentiation and long-term depression of synaptic ef®cacy. To develop Caenorhabditis elegans as a model system for studying the neuronal function of the cGKs, we cloned and characterized the cgk-1 gene. A combination of approaches showed that cgk-1 produces three transcripts, which differ in their ®rst exon but are similar in length. Northern analysis of C. elegans RNA, performed with a probe designed to hybridize to all three transcripts, con®rmed that a major 3.0 kb cgk-1 transcript is present at all stages of development. To determine if the CGK-1C protein was a cGMP-dependent protein kinase, CGK-1C was expressed in Sf9 cells and puri®ed. CGK-1C shows a K a of 190^14 nM for cGMP and 18.4^2 mM for cAMP. Furthermore, CGK-1C undergoes autophosphorylation in a cGMP-dependent manner and is inhibited by the commonly used cGK inhibitor, KT5823. To determine which cells expressed CGK-1C, a 2.4-kb DNA fragment from the promoter of CGK-1C was used to drive GFP expression. The CGK-1C reporter construct is strongly expressed in the ventral nerve cord and in several other neurons as well as the marginal cells of the pharynx and intestine. Finally, RNAmediated interference of CGK-1 resulted in movement defects in nematode larvae. These results provide the ®rst demonstration that cGMP-dependent protein kinase is present in neurons of C.elegans and show that this kinase is required for normal motility.

Substantial guanylyl cyclase activity was detected in membrane fractions prepared from Caenorhabditis elegans (100 pmol cGMP/min/mg at 20°C or 500 pmol cGMP/min/mg at 37°C), suggesting the potential existence of orphan cyclase receptors in the nematode. Using degenerate primers, a cDNA clone encoding a putative membrane form of the enzyme (GCY-X 1 ) was obtained. The apparent cyclase was most closely related to the mammalian natriuretic peptide receptor family, and retained cysteine residues conserved within the extracellular domain of the mammalian receptors. Expression of the cDNA in COS-7 cells resulted in low, but detectable guanylyl cyclase activity (about 2-fold above vector alone). The extracellular and protein kinase homology domain of the mammalian receptor (GC-B) for C-type natriuretic peptide (CNP) was fused to the catalytic domain of GCY-X 1 and expressed in COS-7 cells to determine whether ligand-dependent regulation would now be obtained. The resulting chimeric protein (GC-BX 1 ) was active, and CNP elevated cGMP in a concentrationdependent manner. Subsequently, a search of the genome data base demonstrated the existence of at least 29 different genes from C. elegans that align closely with the catalytic domain of GCY-X 1 , and thus an equally large number of different regulatory ligands may exist. Guanylyl cyclases appear to fall into two general families. (i)The membrane forms contain an extracellular ligand binding domain, a single transmembrane segment, and an intracellular protein kinase-like and cyclase catalytic domain; and (ii) the soluble forms contain two heterologous subunits, both of which contain a consensus cyclase catalytic domain (1, 2). These general features of the soluble and membrane forms appear to be conserved across many species, although expression of various invertebrate cDNAs encoding putative membrane guanylyl cyclases in mammalian cultured cells has not resulted in detectable enzyme activity (3-5).Caenorhabditis elegans has proven a highly effective animal model for unraveling the functions of specific signaling pathways, the power resting particularly in the well defined lineage of each cell and the use of genetics. While cGMP has been measured (6) and a cGMP-dependent protein kinase has been purified from nematodes (7), C. elegans has been largely ignored as a means to define the functions of the various guanylyl cyclase/cGMP signaling pathways even though recent work on disruption of the cyclic nucleotide-gated ion channel has resulted in defective sensory neuron function (8,9).In preliminary studies on crude membrane fractions from C. elegans, guanylyl cyclase activity was easily measured, and PCR 1 subsequently resulted in the identification of a cDNA clone encoding a putative guanylyl cyclase that was most closely related to the mammalian natriuretic peptide receptor family. The conservation of cysteine residues within the extracellular domain when compared with the mammalian natriuretic peptide receptors also suggested that the putative cyclase represented an orph...