Abstract
Transmembrane receptors, a subset of integral membrane proteins, are the receivers that transduce an extracellular chemical message into an intracellular response. Accordingly, these proteins are of particular interest in the scientific community and are probably best studied as part of a cellular system. Herein, we detail the engineering of a fluorescent and bioluminescent reporter cell line for a transmembrane receptor and how to employ it in a directed evolution screen that identifies peptide regulators of receptor activity.
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References
von Heijne G (2007) The membrane protein universe: what’s out there and why bother? J Intern Med 261(6):543–557
Lappano R, Maggiolini M (2011) G protein-coupled receptors: novel targets for drug discovery in cancer. Nat Rev Drug Discov 10(1): 47–60
Overington JP, Al-Lazikani B, Hopkins AL (2006) How many drug targets are there? Nat Rev Drug Discov 5(12):993–996
Russ AP, Lampel S (2005) The druggable genome: an update. Drug Discov Today 10(23–24):1607–1610
Katzen F, Peterson TC, Kudlicki W (2009) Membrane protein expression: no cells required. Trends Biotechnol 27(8):455–460
Maurice P et al (2011) GPCR-interacting proteins, major players of GPCR function. Adv Pharmacol 62:349–380
Akira S (2003) Toll-like receptor signaling. J Biol Chem 278(40):38105–38108
Daugherty PS, Iverson BL, Georgiou G (2000) Flow cytometric screening of cell-based libraries. J Immunol Methods 243(1–2): 211–227
Arnold FH (1998) Design by directed evolution. Accounts Chem Res 31:125–131
Cammett TJ et al (2010) Construction and genetic selection of small transmembrane proteins that activate the human erythropoietin receptor. Proc Natl Acad Sci USA 107(8):3447–3452
Caputo GA et al (2008) Computationally designed peptide inhibitors of protein-protein interactions in membranes. Biochemistry 47(33):8600–8606
Chin CN, Sachs JN, Engelman DM (2005) Transmembrane homodimerization of receptor-like protein tyrosine phosphatases. FEBS Lett 579(17):3855–3858
Finger C, Escher C, Schneider D (2009) The single transmembrane domains of human receptor tyrosine kinases encode self-interactions. Sci Signal 2(89):ra56
Li R et al (2004) Dimerization of the transmembrane domain of Integrin alphaIIb subunit in cell membranes. J Biol Chem 279(25): 26666–26673
Nemoto W, Toh H (2006) Membrane interactive alpha-helices in GPCRs as a novel drug target. Curr Protein Pept Sci 7(6):561–575
Yin H et al (2007) Computational design of peptides that target transmembrane helices. Science 315(5820):1817–1822
Zhang H et al (2002) Integrin-nucleated Toll-like receptor (TLR) dimerization reveals subcellular targeting of TLRs and distinct mechanisms of TLR4 activation and signaling. FEBS Lett 532(1–2):171–176
Zhu H et al (2010) Specificity for homooligomer versus heterooligomer formation in integrin transmembrane helices. J Mol Biol 401(5): 882–891
Talbert-Slagle K et al (2009) Artificial transmembrane oncoproteins smaller than the bovine papillomavirus E5 protein redefine sequence requirements for activation of the platelet-derived growth factor beta receptor. J Virol 83(19):9773–9785
Horwitz BH et al (1988) 44-amino-acid E5 transforming protein of bovine papillomavirus requires a hydrophobic core and specific carboxyl-terminal amino acids. Mol Cell Biol 8(10):4071–4078
Klein O et al (1999) The bovine papillomavirus E5 protein requires a juxtamembrane negative charge for activation of the platelet-derived growth factor beta receptor and transformation of C127 cells. J Virol 73(4):3264–3272
Klein O et al (1998) Role of glutamine 17 of the bovine papillomavirus E5 protein in platelet-derived growth factor beta receptor activation and cell transformation. J Virol 72(11):8921–8932
Schlegel R et al (1986) The E5 transforming gene of bovine papillomavirus encodes a small, hydrophobic polypeptide. Science 233(4762):464–467
Sparkowski J et al (1996) E5 oncoprotein transmembrane mutants dissociate fibroblast transforming activity from 16-kilodalton protein binding and platelet-derived growth factor receptor binding and phosphorylation. J Virol 70(4):2420–2430
Surti T et al (1998) Structural models of the bovine papillomavirus E5 protein. Proteins 33(4):601–612
Marlatt SA et al (2011) Construction and maintenance of randomized retroviral expression libraries for transmembrane protein engineering. Protein Eng Des Sel 24(3): 311–320
Acknowledgements
We thank the National Science Foundation (CHE 0954819) and the National Institutes of Health (GM 103843 and GM 101279) for financial supports.
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Lluis, M.W., Yin, H. (2013). Engineering and Utilization of Reporter Cell Lines for Cell-Based Assays of Transmembrane Receptors. In: Ghirlanda, G., Senes, A. (eds) Membrane Proteins. Methods in Molecular Biology, vol 1063. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-583-5_12
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DOI: https://doi.org/10.1007/978-1-62703-583-5_12
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