The Escherichia coli chemoreceptors for serine (Tsr) and aspartate (Tar) and several bacterial class III adenylyl cyclases (ACs) share a common molecular architecture; that is, a membrane anchor that is linked via a cytoplasmic HAMP domain to a C-terminal signal output unit. Functionality of both proteins requires homodimerization. The chemotaxis receptors are well characterized, whereas the typical hexahelical membrane anchor (6TM) of class III ACs, suggested to operate as a channel or transporter, has no known function beyond a membrane anchor. We joined the intramolecular networks of Tsr or Tar and two bacterial ACs, Rv3645 from Mycobacterium tuberculosis and CyaG from Arthrospira platensis, across their signal transmission sites, connecting the chemotaxis receptors via different HAMP domains to the catalytic AC domains. AC activity in the chimeras was inhibited by micromolar concentrations of L-serine or L-aspartate in vitro and in vivo. Single point mutations known to abolish ligand binding in Tar (R69E or T154I) or Tsr (R69E or T156K) abrogated AC regulation. Co-expression of mutant pairs, which functionally complement each other, restored regulation in vitro and in vivo. Taken together, these studies demonstrate chemotaxis receptor-mediated regulation of chimeric bacterial ACs and connect chemical sensing and AC regulation.The canonical chemotaxis receptors Tsr for serine and Tar for aspartate of Escherichia coli have been studied extensively as model signaling systems that regulate bacterial swimming behavior (1-5). Ligand binding to the periplasmic domain of the receptor initiates a biochemically defined cascade of events that finally results in reversal of the flagellar beat (4, 6). The chemotaxis receptors are tripartite proteins with a periplasmic receptor anchored in the cytoplasmic membrane by two transmembrane spans followed by a HAMP domain (histidine kinases, adenylyl cyclases, methyl-accepting proteins of chemotaxis, and protein phosphatases (7)) which adjoins the second transmembrane helix and serves as a signal converter followed by an output domain as C terminus (3). Homodimerization of Tsr or Tar chemoreceptors is required for function, and the ligands bind in the periplasmic interface between the subunits (8 -11). The ubiquitous HAMP domains are signal converters of 55-60 amino acids with a homodimeric fourhelical parallel coiled-coil conformation (12). In the chemotaxis receptors the HAMP domain operates as a C-terminal histidine kinase control unit that has no known intrinsic enzymatic activity. As this molecular architecture is predominant in the 30 known sensor kinases of E. coli, a variety of chimeric sensor proteins has been generated successfully in the past that consist of modules derived from different E. coli sensors (see examples in Refs. 13-15).A similar tripartite domain architecture as in chemotaxis receptors exists in several class III AC 2 isoforms in eubacteria. Class III AC isoforms outnumber all others, are prevalent in bacteria, and include all eukaryotic ACs (16 -17). ...

A long-sought molecule (1) was recently identified as acting independent of COX-1/-2 (2). The potent analgesic and anti-pyretic actions of acetaminophen lacking anti-inflammatory potency suggested the presence of an additional cyclo-oxygenase that could be directly responsible for acetaminophen-sensitive generation of prostanoids in neuronal systems. This could now be understood at least in part as modulation of the recently identified COX-3 (2). In canine, alternative splicing generates four different mRNA variants derived from the COX-1 geneOCOX-3, COX-1, and two partial COX-1 (denoted as PCOX-1 proteins), PCOX-1a and PCOX-1b, encoding novel members of the COX-1 protein family. Among them only canine COX-1 and canine COX-3 possessed glycosylation-dependent cyclooxygenase activity. The discovery by Dan Simmons and colleagues of COX-3 will most surely herald more interest in these pathways and enzymes as recently illustrated by the number, scholarship, and import of several commentaries (3-6).Predicted canine COX-3 is composed of COX-1 and the retained intron 1 (2). Identification of canine COX-3 might have a significant effect on understanding inflammatory messengers, characterizing new routes of prostanoid formation and pain conduction (3-6). These findings may be crucial to the development of new therapeutic agents targeting both COX-2 and COX-3 in order to reduce CNS inflammatory reactions, fever, and pain. However, this may hold true only for the dog. Database analysis of human COX-1 (Fig. 1) showed a frameshift induced by intron 1, possibly revealing COX-3 to be a virtual protein in humans. However, Western blot analysis of human aorta tissue using polyclonal antibodies directed against the first 13 amino acids of the predicted human and dog/mouse COX-3 detected (surprisingly) a 65 kDa protein postulated to be human COX-3 (2).The high sequence similarity of the canine insert to intron 1 of either human or mouse COX-1 genes and the presence of 5Ј and 3Ј consensus splice sites indicated the complete retention of intron 1 in this transcript. The predicted retention of a complete intron 1 sequence in human COX-1 (OMIM accession #176805) from a screening of several databases revealed a frameshift due to insertion of intron 1 consisting of 94 nucleotides or 98 nucleotides in the murine COX-1 gene, respectively. The frameshift after the insert (see Fig. 2) results 1) in a TGA stop codon 48 amino acids later and 2) a completely changed protein sequence not related to human COX-1. The compensating molecular events suggested by Chandrasekharan et al. are unlikely. COX-1 polymorphisms or sequencing errors concerning the nucleotides of intron 1 are implausible since recent screening of the human COX-1 gene, including the complete intron 1 region, revealed no polymorphisms or deletions in intron 1 (7). Moreover, compensation by "-1" ribosomal frame shifting has not been reported in eukaryotic mRNAs to date. COX-3 is considered a new and important lead in the generation of anti-inflammatory and analgesic agents. However,...

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