Cloning and characterization of a novel class I cytokine receptor.

The human gp130 cDNA sequence was used as a query to search an expressed sequence tag database (dbEST) to identify cDNA sequences with similarity to the cytokine class I receptor family. A novel class I cytokine receptor was identified in a human infant brain cDNA library and was named WSX-1. Full-length cDNA sequences for human and murine WSX-1 were isolated and characterized. The WSX-1 cDNA encodes a 636 amino acid transmembrane protein with an extracellular domain of 482 amino acids and a cytoplasmic domain of 96 amino acids. The structure of the WSX-1 protein most closely resembles that of gp130. Northern blot analysis indicates high levels of expression in thymus, spleen, lymph node, and peripheral blood leukocytes, suggesting a role for WSX-1 in modulation of the immune response.

High- and low-abundance chemoreceptors in Escherichia coli: differential activities associated with closely related cytoplasmic domains.

In Escherichia coli, two high-abundance chemoreceptors are present in cellular dosages approximately ten-fold greater than two low-abundance receptors. In the absence of high-abundance receptors, cells exhibit an abnormally low tumble frequency and the ability of the remaining receptors to mediate directed migration in spatial gradients is substantially compromised. We found that increasing the cellular amount of the low-abundance receptor Trg over a range of dosages did not alleviate these defects and thus concluded that high- and low-abundance receptors are distinguished not simply by their different dosages in a wild-type cell but also by an inherent difference in activity. By creating hybrids of the low-abundance receptor Trg and the high-abundance receptor Tsr, we investigated the possibility that this inherent difference could be localized to a specific receptor domain and found that the cytoplasmic domain of the high-abundance receptor Tsr conferred the essential features of that receptor class on the low-abundance receptor Trg, even though it is in this domain that residue identity between the two receptors is substantially conserved.

Mutational analysis of a transmembrane segment in a bacterial chemoreceptor.

Trg is a member of a family of receptors that mediates chemotaxis by Escherichia coli. Its transmembrane domain is a loose four-helix bundle consisting of two helices from each of the two identical subunits. This domain mediates transmembrane signaling through a conformational change in which the second transmembrane segment (TM2) is thought to move relative to TM1, but mutational analysis of TM2 by cysteine scanning had identified only a few positions at which substitutions perturbed function or induced signaling. Thus, we performed mutational analysis by random mutagenesis and screening. Among 42 single-residue substitutions in TM2 that detectably altered function, 16 had drastic effects on receptor activity. These substitutions defined a helical face of TM2. This functionally important surface was directed into the protein interior of the transmembrane domain, where TM2 faces the helices or the other subunit. The functionally perturbing substitutions did not appear to cause general disruption of receptor structure but rather had more specific effects, altering aspects of transmembrane signaling. An in vivo assay of signaling identified some substitutions that reduced and others that induced signaling. These two classes were distributed along adjacent helical faces in a pattern that strongly supports the notion that conformational signaling involves movement between TM2 and TM1 and that signaling is optimal when stable interactions are maintained across the interface between the homologous helices in the transmembrane domain. Our mutational analysis also revealed a striking tolerance of the chemoreceptor for substitutions, including charged residues, usually considered to be disruptive of transmembrane segments.

The role of the WSXWS equivalent motif in growth hormone receptor function.

Members of the cytokine receptor family have a consensus WSXWS sequence (WS motif) in the extracellular domain. With the interleukin-2, erythropoietin, and prolactin receptors, alteration of the WS sequence disrupts ligand binding and receptor signaling. The structural basis for these effects is unclear. To examine the role of the WS equivalent sequence (Y222GEFS226) in the function of the growth hormone receptor, each residue was mutated to alanine or to the WS consensus sequence. Although we used stable cell lines expressing all of these mutants, we show only three mutants, Y222A, G223A, and S226A, which display lower ligand affinity. Using conformation-specific monoclonal antibodies, we show that Y222A and S226A receptors have structural perturbations, which result in decreased signal transduction. This was shown by a decreased ability of growth hormone to stimulate protein synthesis and to transactivate the c-fos promoter with these mutants. The crystal structure of the ligand-occupied extracellular domain of growth hormone receptor indicates that Tyr222 and Ser226 have important interactions within the second beta-barrel domain, providing a structural basis for our results. The WS segment is not involved in sequence-specific accessory protein interaction, as mutation of residues Gly223, Glu224, and Phe225 does not alter receptor function.

Transmembrane signalling by a hybrid protein: communication from the domain of chemoreceptor Trg that recognizes sugar-binding proteins to the kinase/phosphatase domain of osmosensor EnvZ.

Chemoreceptor Trg and osmosensor EnvZ of Escherichia coli share a common transmembrane organization but have essentially unrelated primary structures. We created a hybrid gene coding for a protein in which Trg contributed its periplasmic and transmembrane domains as well as a short cytoplasmic segment and EnvZ contributed its cytoplasmic kinase/phosphatase domain. Trz1 transduced recognition of sugar-occupied, ribose-binding protein by its periplasmic domain into activation of its cytoplasmic kinase/phosphatase domain as assessed in vivo by using an ompC-lacZ fusion gene. Functional coupling of sugar-binding protein recognition to kinase/phosphatase activity indicates shared features of intramolecular signalling in the two parent proteins. In combination with previous documentation of transduction of aspartate recognition by an analogous fusion protein created from chemoreceptor Tar and EnvZ, the data indicate a common mechanism of transmembrane signal transduction by chemoreceptors and EnvZ. Signalling through the fusion proteins implies functional interaction between heterologous domains, but the minimal sequence identity among relevant segments of EnvZ, Tar, and Trg indicates that the link does not require extensive, specific interactions among side chains. The few positions of identity in those three sequences cluster in transmembrane segment 1 and the short chemoreceptor sequence in the cytoplasmic part of the hybrid proteins. These regions may be particularly important in physical and functional coupling. The specific cellular conditions necessary to observe ligand-dependent activation of Trz1 can be understood in the context of the importance of phosphatase control in EnvZ signalling and limitations on maximal receptor occupancy in binding protein-mediated recognition.

Proteins antigenically related to methyl-accepting chemotaxis proteins of Escherichia coli detected in a wide range of bacterial species.

The four methyl-accepting chemotaxis proteins of Escherichia coli, often called transducers, are transmembrane receptor proteins that exhibit substantial identity among the sequences of their cytoplasmic domains. Thus, antiserum raised to one of these proteins recognizes the others and might be expected to recognize related proteins in other bacteria. We used antiserum raised to the transducer Trg in immunoblot experiments to probe a wide range of bacterial species for the presence of antigenically related proteins. Such proteins were detected in over 20 different species, representing 6 of the 11 eubacterial phyla defined by analysis of rRNA sequences as well as one archaebacterial group. Species containing proteins antigenically related to the transducers of E. coli included members of all four subdivisions of the phylum in which E. coli is placed, members of four of the six subdivisions of spirochetes, and two gliding bacteria. These observations provide substantial support for the notion that methyl-accepting taxis proteins are widely distributed among the diversity of bacterial species.