In Escherichia coli, two high-abundance chemoreceptors are present in cellular dosages approximately tenfold 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 lowabundance receptor Trg over a range of dosages did not alleviate these defects and thus concluded that highand 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 highabundance 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.Methyl-accepting chemoreceptor proteins mediate the chemotactic response of Escherichia coli to an array of attractants and repellents (5, 9, 10). Related proteins have been detected in a substantial number of bacterial species (20), and it appears that such proteins are generally components of the sensory systems that direct motile bacteria to favorable environments. The hallmarks of this chemoreceptor family are a highly conserved region (ϳ40 residues) involved in intracellular signaling and adjoining conserved regions containing methyl-accepting glutamyl residues that are covalently modified in the process of sensory adaptation. Both of these regions are located in the carboxyl-terminal half of the chemoreceptor. Studies of chemoreceptors from E. coli and Salmonella typhimurium have implicated the involvement of the highly conserved region in the control of a noncovalently associated histidine kinase, CheA (5, 25), and have identified two distinct actions: (i) basal activation of the kinase and (ii) modulation of kinase activity in response to a change in receptor occupancy. Basal activation, effected by unoccupied or adapted receptors, is in essence a steady-state signaling that determines the cellular content of the phosphorylated response regulator CheY (phosphoCheY). Phospho-CheY interacts with the flagellar switch to induce clockwise (CW) rotation by the otherwise counterclockwise (CCW)-rotating motor. In a wild-type cell, basal activation creates a level of phospho-CheY that results in a balance between CCW and CW flagellar rotation that creates a corresponding alternation between smooth swimming and tumbling. This alternation causes the cell to trace a random walk. Modulation of kinase activity from its level of basal activation occurs through receptors that have undergone a change in receptor occupancy but have not yet adapted. It alters the content of phospho-CheY, an...
Galanin-like peptide (GALP) shares sequence homology with galanin and binds to galanin receptors in vitro. GALP neurons in the arcuate nucleus coexpress leptin receptors, and GALP mRNA expression is up-regulated by leptin. Based on these observations, we postulated that GALP plays a role in mediating leptin's inhibitory effects on food intake (FI) and body weight (BW), as well as its stimulatory effect on the reproductive axis. To test these hypotheses, we performed several studies in which mice received intracerebroventricular injections of either GALP or vehicle. Acute GALP treatment elicited a dose-dependent suppression of FI and BW. Long-term treatment with GALP caused only transient reductions in FI and BW, demonstrating that the mice became refractory to continued exposure to GALP. GALP inhibited FI as early as 1 h post injection. Central injection of GALP suppressed locomotor activity and elicited the formation of a conditioned taste aversion. In male mice, serum levels of LH and testosterone were increased by GALP administration. Although we cannot rule out possible nonspecific effects of GALP on FI, the present observations are consistent with the argument that GALP is a downstream effector of leptin's actions within the central nervous system.
Fig. 1. The construction of pRB020 required that pGB1 be treated to remove its sole NdeI site. This was accomplished by digestion with that endonuclease and then treatment with mung bean nuclease and ligation. Elimination of the NdeI site, which is located outside of the genes carried on the plasmid, did not have a discernible effect on the LacI-mediated expression of trg. The pGB1 derivative lacking the NdeI site was cleaved with AflIl and StuI, and the resulting 0.7-kb fragment of trg was replaced by one in which an NdeI site had been previously introduced at codons 266 and 267 by oligonucleotide-directed mutagenesis, to create pJB34. Plasmids pDR200 (8) and pJB34 were digested with Hindlll and NdeI, and the 1-kb fragment of the former was joined to the 7-kb fragment of the latter to yield pRB020.Methods. The immunoblot procedures used were described by Morgan et al. (26) and used antiserum raised to purified Trg or purified EnvZ, anti-rabbit immunoglobulin G raised in goats and coupled to horseradish peroxidase, and 1-chloro-4-naphthol as a chromogenic substrate for the peroxidase. The assay of 3-galactosidase and the units of activity used were described by Miller (24). RESULTSConstruction of a trg-envZ hybrid. We aimed to create a hybrid gene between trg and envZ that coded for a chimeric protein analogous to the Tazl protein produced by the tarenvZ hybrid (33). The hybrid gene and fusion protein are diagrammed in Fig. 1 (Fig. 2B) as the result of a flhD mutation. Production of Trz was assessed by immunoblot analysis using anti-Trg and anti-EnvZ (Fig. 2). In IPTG-induced cells, a band of approximately 54 kDa was recognized by both antisera. We concluded that this band represented Trzl because its size corresponded to the predicted value of 54.5 kDa, it was recognized by antisera to Trg and to EnvZ, and induction of the promoter controlling trz] expression substantially increased its cellular content.
Galanin and its newly discovered relative galanin-like peptide (GALP) are neuropeptides that are implicated in the neuroendocrine regulation of body weight and reproduction. GALP has been shown to bind in vitro to galanin receptor subtypes 1 and 2, but whether it has its own specific receptor(s) is unknown. We reasoned that if GALP acts through a receptor that is distinct from galanin receptors, then GALP should activate central pathways that are different from those activated by galanin. The purpose of this study was to determine whether galanin and GALP produce different patterns of neuronal activation within the hypothalamus. Quantitative analysis of Fos immunoreactivity showed that galanin induced a significantly greater number of Fos-positive nuclei in the paraventricular nucleus compared with GALP (P < 0.001); however, compared with galanin, GALP induced significantly more Fos-positive cells in the horizontal limb of the diagonal band of Broca, caudal preoptic area, arcuate nucleus, and median eminence (P < 0.05). These observations suggest that GALP and galanin act through different receptor-mediated pathways to exert their effects on the regulation of body weight and reproduction and identify target cells for GALP's specific actions in the hypothalamus, including the preoptic area, paraventricular and arcuate nuclei, and the median eminence.
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