DNA Display Selection of Peptide Ligands for a Full-Length Human G Protein-Coupled Receptor on CHO-K1 Cells

The G protein-coupled receptors (GPCRs), which form the largest group of transmembrane proteins involved in signal transduction, are major targets of currently available drugs. Thus, the search for cognate and surrogate peptide ligands for GPCRs is of both basic and therapeutic interest. Here we describe the application of an in vitro DNA display technology to screening libraries of peptide ligands for full-length GPCRs expressed on whole cells. We used human angiotensin II (Ang II) type-1 receptor (hAT1R) as a model GPCR. Under improved selection conditions using hAT1R-expressing Chinese hamster ovary (CHO)-K1 cells as bait, we confirmed that Ang II gene could be enriched more than 10,000-fold after four rounds of selection. Further, we successfully selected diverse Ang II-like peptides from randomized peptide libraries. The results provide more precise information on the sequence-function relationships of hAT1R ligands than can be obtained by conventional alanine-scanning mutagenesis. Completely in vitro DNA display can overcome the limitations of current display technologies and is expected to prove widely useful for screening diverse libraries of mutant peptide and protein ligands for receptors that can be expressed functionally on the surface of CHO-K1 cells

Amino acid sequences of selected clones from the randomized library-I.

<p>(<b>A</b>) The designed sequence is XRΩYΩXΩF where X = F, L, I, M, V, S, P, T, A, Y, H, Q, N, K, D, E, C, W, R or G; and Ω = F, L, I, M, V, S, P, T or A. Fixed residues are shown in blue, and conserved residues are red. The number of clones containing each sequence is indicated. Sequences of seven clones with a frameshift in the preceding linker region are not shown. The binding activity (+, specific binding; +/−, nonspecific binding; −, no binding) of each peptide is also shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030084#pone-0030084-g006" target="_blank">Figures 6</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030084#pone-0030084-g007" target="_blank">7</a>. IC₅₀ was determined by means of competitive binding assays at various concentrations of synthetic peptides (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030084#s4" target="_blank">MATERIALS AND METHODS</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030084#pone.0030084.s001" target="_blank">Figure S1</a>). (<b>B</b>) Sequence logos representation <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030084#pone.0030084-Schneider1" target="_blank">[45]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030084#pone.0030084-Crooks1" target="_blank">[46]</a> of the peptide sequences with specific binding activity (+). The height of each column reflects the bias from random of particular residues. Fixed residues are shown in gray. Polar amino acids containing an amide group (Q, N) and the rest of them are shown in purple and green, respectively, basic charged residues in deep blue, and hydrophobic residues in black.</p

Effect of selected peptides on the concentration of calcium in the hAT1R-expressing cells.

<p>Fura-2/AM-loaded hAT1R/CHO-K1 or Mock/CHO-K1 cells were exposed to synthetic octapeptides (<b>A</b>) LI5-1 (10 nM), (<b>B</b>) LI5-2 (10 nM), (<b>C</b>) LII3-2 (10 nM), (<b>D</b>) LII3-4 (100 nM), and (<b>E</b>) LII3-5 (1 µM) and/or Ang II (1 nM), respectively, as indicated by the arrows. The results are expressed as Fura-2 fluorescence ratio (340/380 nm).</p

Binding assays of selected peptides from the randomized libraries with the hAT1R-expressing cells.

<p>Selected streptavidin-fused peptides were generated by the PURE system. An aliquot of the reaction mixture was incubated with the hAT1R/CHO-K1 cells (hAT1R +) or the Mock/CHO-K1 cells (hAT1R −). The cells were washed, and the bound molecules were detected with anti-T7·tag antibody. The sequences of the peptides are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030084#pone-0030084-g004" target="_blank">Figures 4A</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030084#pone-0030084-g005" target="_blank">5A</a>.</p

Stable expression of a model GPCR, the recombinant human angiotensin II type 1 receptor (hAT1R) with the C-terminal c-myc tag, on CHO-K1 cells.

<p>(<b>A</b>) Immunofluorescence staining of hAT1R-c-myc-expressing CHO-K1 cells (hAT1R/CHO-K1, <b>left</b>) and CHO-K1 cells transfected with the empty vector alone (Mock/CHO-K1, <b>right</b>) with anti-c-myc antibody (green). Nuclei were stained with PI (red). (<b>B</b>) Confirmation of the function of hAT1R-c-myc by monitoring changes in intracellular Ca²⁺ in response to angiotensin II (Ang II). Fura-2/AM-loaded hAT1R/CHO-K1 or Mock/CHO-K1 cells were exposed to 1 nM Ang II at the point indicated by the arrow. The results are expressed as Fura-2 fluorescence ratio (340/380 nm).</p

Construction and enrichment of the streptavidin-fused angiotensin II (STA-Ang II) gene in multiple rounds of DNA-display selection on hAT1R/CHO-K1 cells.

<p>(<b>A</b>) A schematic representation of the DNA template for <i>in vitro</i> transcription/translation. DNA was labeled during PCR with photocleavable biotin <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030084#pone.0030084-Doi3" target="_blank">[22]</a> at the upstream ends and with fluorescein at the downstream ends, using labeled primers. The translated open reading frame consists of sequences for a T7·tag, streptavidin (STA), a peptide linker, and Ang II gene. The 5′-UTR fragment contains T7 promoter. (<b>B</b>) Reaction mixtures containing 1∶100 or 1∶10,000 molar ratio of STA-Ang II: STA genes were emulsified. The DNA after each round of selection was PCR-amplified with a fluorescein-labeled primer and analyzed by 15% PAGE with an imaging analyzer.</p