The Tar chemoreceptor of Escherichia coli is a membrane-bound sensory protein that facilitates bacterial chemotaxis in response to aspartate. The EnvZ molecule has a membrane topology similar to Tar and is a putative osmosensor that is required for osmoregulation of the genes for the major outer membrane porin proteins, OmpF and OmpC. The cytoplasmic signaling domain of Tar was replaced with the carboxyl portion of EnvZ, and the resulting chimeric receptor activated transcription of the ompC gene in response to aspartate. The activation of ompC by the chimeric receptor was absolutely dependent on OmpR, a transcriptional activator for ompF and ompC.

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.

Insulin is thought to elicit its effects by crosslinking the two extracellular ␣-subunits of its receptor, thereby inducing a conformational change in the receptor, which activates the intracellular tyrosine kinase signaling cascade. Previously we identified a series of peptides binding to two discrete hotspots on the insulin receptor. Here we show that covalent linkage of such peptides into homodimers or heterodimers results in insulin agonists or antagonists, depending on how the peptides are linked. An optimized agonist has been shown, both in vitro and in vivo, to have a potency close to that of insulin itself. The ability to construct such peptide derivatives may offer a path for developing agonists or antagonists for treatment of a wide variety of diseases.I nsulin is one of the most studied peptide hormones because of its importance in maintaining glucose homeostasis. This 51-aa hormone is very well characterized with regard to its structure, both in crystal form and in solution. The insulin receptor (IR) is a transmembrane ␣ 2 ␤ 2 glycoprotein whose intracellular tyrosine kinase domain is activated by binding of insulin, leading to a cascade of intracellular signaling events. The kinase domain of the IR (1) and an extracellular fragment of the related receptor for insulin-like growth factor I (IGF-IR; ref. 2) have been crystallized, but the structure of the insulin binding domain of the IR is not known, and the mechanism for the transmission of a signal through its transmembrane domain is not well understood. A model for the binding and activation has been proposed in which insulin uses two different sites on its surface to crosslink the two ␣-subunits of the IR, thus inducing a conformational change that activates the receptor (refs. 3 and 4; Fig. 1).In a previous report (5), we panned random, highly diverse peptide display libraries against the IR. By using this approach, we identified a large number of peptides binding to the IR and competing for insulin binding with micromolar or submicromolar affinity, although these peptides had no sequence homology with insulin. These peptides bound to two discrete hotspots on the receptor (designated site 1 and site 2), and these hotspots appeared to correspond to the two contact sites involved in insulin binding predicted by the crosslinking model (ref. 3 and J.B., unpublished results). At least two different sequence motifs were found for site 1 peptides, and some of these were full agonists but of low affinity. Other site 1 peptides were antagonists, whereas site 2 peptides were either antagonists or inactive. The mechanism behind the agonism of the site 1 peptides is not known, but it has been speculated that site 1 binding may be important for receptor activation, whereas the role of the site 2 interaction may be more related to affinity and selectivity. In addition to these two families of peptides, a third group was identified, but no further work has been done on this group. In the present work, we have used site 1 and site 2 peptides as building blocks ...

We used phage display to generate surrogate peptides that define the hotspots involved in protein-protein interaction between insulin and the insulin receptor. All of the peptides competed for insulin binding and had affinity constants in the high nanomolar to low micromolar range. Based on competition studies, peptides were grouped into non-overlapping Sites 1, 2, or 3. Some Site 1 peptides were able to activate the tyrosine kinase activity of the insulin receptor and act as agonists in the insulin-dependent fat cell assay, suggesting that Site 1 marks the hotspot involved in insulin-induced activation of the insulin receptor. On the other hand, Site 2 and 3 peptides were found to act as antagonists in the phosphorylation and fat cell assays. These data show that a peptide display can be used to define the molecular architecture of a receptor and to identify the critical regions required for biological activity in a site-directed manner.

A library of long peptides displayed on the pIII protein of filamentous phage was used in biopanning experiments against several protein targets. We find that a large percentage of phage clones that bind specifically to a target contain peptide-encoding genes that do not have an ORF. Instead, the reading frame is either interrupted by one or more nonsuppressed stop codons, or a post-transcriptional frameshift is needed to account for the expression of the minor phage coat protein pIII. The percentage of frameshifted clones varies depending on the target. It can be as high as 90% for clones specific for soluble forms of certain cytokine receptors. Conversely, biopanning against four mAbs did not yield any frameshifted clones. Our studies focused on one clone that binds specifically to rat growth hormone binding protein (GHBP) yet does not have an ORF. A secondary peptide library containing random mutations of this sequence was constructed and panned against GHBP to optimize and correct the reading frame. In the last round (round two) of panning with this library, none of the phage clones that bound to GHBP had an ORF. However, careful analysis of these clones allowed us to design a synthetic peptide capable of binding to GHBP. The results of this study indicate that ORFs are not required to obtain gene expression of the minor coat protein of filamentous phage and suggest that some ORF ؊ clones may have a selective advantage over the clones having ORFs.