A Microtiter Assay for Quantifying Protein-Protein Interactions Associated with Cell-Cell Adhesion

Abstract

Cell-cell adhesions are a hallmark of epithelial tissues, and the disruption of these contacts plays a critical role in both the early and late stages of oncogenesis. The interaction between the transmembrane protein E-cadherin and the intracellular protein β-catenin plays a crucial role in the formation and maintenance of epithelial cell-cell contacts and is known to be downregulated in many cancers. The authors have developed a protein complex enzyme-linked immunosorbent assay (ELISA) that can quantify the amount of β-catenin bound to E-cadherin in unpurified whole-cell lysates with a Z′ factor of 0.74. The quantitative nature of the E-cadherin:β-catenin ELISA represents a dramatic improvement over the low-throughput assays currently used to characterize endogenous E-cadherin:β-catenin complexes. In addition, the protein complex ELISA format is compatible with standard sandwich ELISAs for parallel measurements of total levels of endogenous E-cadherin and β-catenin. In 2 case studies closely related to cancer cell biology, the authors use the protein complex ELISA and traditional sandwich ELISAs to provide a detailed, quantitative picture of the molecular changes occurring within adherens junctions in vivo. Because the E-cadherin: β-catenin protein complex plays a crucial role in oncogenesis, this protein complex ELISA may prove to be a valuable quantitative prognostic marker of tumor progression. (Journal of Biomolecular Screening 2007:683-693)

Keywords

E-cadherin β-catenin cell-cell adhesion cancer ELISA

References

Wijnhoven BP , Dinjens WN , Pignatelli M. : E-cadherin-catenin cell-cell adhesion complex and human cancer. Br J Surg 2000;87:992-1005.

Christofori G. , Semb H. : The role of cell-adhesion molecule E-cadherin as a tumour suppressor gene. Trends Biochem Sci 1999;24:73-76.

Angst BD , Marcozzi C. , Magee AI : The cadherin superfamily. J Cell Sci 2001;114:625-626.

Hirohashi S. : Inactivation of the E-cadherin-mediated cell adhesion system in human cancers. Am J Pathol 1998;153:333-339.

De Leeuw WJ , Berx G. , Vos CB , Peterse JL , Van de Vijver MJ , Litvinov S. , et al: Simultaneous loss of E-cadherin and catenins in invasive lobular breast cancer and lobular carcinoma in situ. J Pathol 1997;183:404-411.

Ohene-Abuakwa Y. , Noda M. , Perenyi M. , Kobayashi N. , Kashima K. , Hattori T. , et al: Expression of the E-cadherin/catenin (α-, β-, and γ-) complex correlates with the macroscopic appearance of early gastric cancer. J Pathol 2000;192:433-439.

Behrens J. , Vakaet L. , Friis R. , Winterhager E. , Van Roy F. , Mareel MM , et al: Loss of epithelial differentiation and gain of invasiveness correlates with tyrosine phosphorylation of the E-cadherin/β-catenin complex in cells transformed with a temperature-sensitive v-SRC gene. J Cell Biol 1993; 120:757-766.

Fujita Y. , Krause G. , Scheffner M. , Zechner D. , Leddy HE , Behrens J. , et al: Hakai, a c-Cbl-like protein, ubiquitinates and induces endocytosis of the E-cadherin complex. Nat Cell Biol 2002 ;4:222-231.

Lu Z. , Ghosh S. , Wang Z. , Hunter T. : Downregulation of caveolin-1 function by EGF leads to the loss of E-cadherin, increased transcriptional activity of βcatenin, and enhanced tumor cell invasion. Cancer Cell 2003;4:499-515.

10.

Avizienyte E. , Wyke AW , Jones RJ , McLean GW , Westhoff MA , Brunton VG , et al: Src-induced de-regulation of E-cadherin in colon cancer cells requires integrin signalling. Nat Cell Biol 2002;4:632-638.

11.

Gottardi CJ , Gumbiner BM : Distinct molecular forms of β-catenin are targeted to adhesive or transcriptional complexes. J Cell Biol 2004;167: 339-349.

12.

Roura S. , Miravet S. , Piedra J. , Garcia de Herreros A. , Dunach M. : Regulation of E-cadherin/catenin association by tyrosine phosphorylation. J Biol Chem 1999;274:36734-36740.

13.

Catimel B. , Layton M. , Church N. , Ross J. , Condron M. , Faux M. , et al: In situ phosphorylation of immobilized receptors on biosensor surfaces: application to E-cadherin/β-catenin interactions. Anal Biochem 2006;357: 277-288.

14.

Kim VN , Mitrophanous K. , Kingsman SM , Kingsman AJ : Minimal requirement for a lentivirus vector based on human immunodeficiency virus type 1. J Virol 1998;72:811-816.

15.

Zhang JH , Chung TD , Oldenburg KR : A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J Biomol Screen 1999;4:67-73.

16.

Wang J. , Qiang H. , Zhang C. , Liu X. , Chen D. , Wang S. : Detection of IgGbound lipoprotein(a) immune complexes in patients with coronary heart disease. Clin Chim Acta 2003;327:115-122.

17.

Strandberg K. , Kjellberg M. , Knebel R. , Lilja H. , Stenflo J. : A sensitive immunochemical assay for measuring the concentration of the activated protein C—protein C inhibitor complex in plasma: use of a catcher antibody specific for the complexed/cleaved form of the inhibitor. Thromb Haemost 2001;86:604-610.

18.

Ogasawara K. , Mashiba S. , Wada Y. , Sahara M. , Uchida K. , Aizawa T. , et al: A serum amyloid A and LDL complex as a new prognostic marker in stable coronary artery disease. Atherosclerosis 2004;174:349-356.

19.

Harris TJ , Peifer M. : Decisions, decisions: β-catenin chooses between adhesion and transcription . Trends Cell Biol 2005;15:234-237.

20.

Butler JE , Navarro P. , Sun J. : Adsorption-induced antigenic changes and their significance in ELISA and immunological disorders. Immunol Invest 1997;26:39-54.

21.

Tait L. , Soule HD , Russo J. : Ultrastructural and immunocytochemical characterization of an immortalized human breast epithelial cell line, MCF-10. Cancer Res 1990;50:6087-6094.

22.

Munemitsu S. , Albert I. , Souza B. , Rubinfeld B. , Polakis P. : Regulation of intracellular β-catenin levels by the adenomatous polyposis coli (APC) tumor-suppressor protein. Proc Natl Acad Sci USA 1995;92:3046-3050.

23.

Korinek V. , Barker N. , Morin PJ , van Wichen D. , de Weger R. , Kinzler KW , et al: Constitutive transcriptional activation by a β-catenin-Tcf complex in APC—/— colon carcinoma. Science 1997;275:1784-1787.

24.

Owens DW , McLean GW , Wyke AW , Paraskeva C. , Parkinson EK , Frame MC , et al: The catalytic activity of the Src family kinases is required to disrupt cadherin-dependent cell-cell contacts. Mol Biol Cell 2000;11:51-64.

25.

Piwnica-Worms H. , Saunders KB , Roberts TM , Smith AE , Cheng SH : Tyrosine phosphorylation regulates the biochemical and biological properties of pp60c-src. Cell 1987;49:75-82.

26.

Cartwright CA , Eckhart W. , Simon S. , Kaplan PL : Cell transformation by pp60c-src mutated in the carboxy-terminal regulatory domain. Cell 1987;49:83-91.

27.

Galliher AJ , Schiemann WP : β3 integrin and Src facilitate transforming growth factor-beta mediated induction of epithelial-mesenchymal transition in mammary epithelial cells. Breast Cancer Res 2006;8:R42.

28.

Hulsken J. , Birchmeier W. , Behrens J. : E-cadherin and APC compete for the interaction with β-catenin and the cytoskeleton. J Cell Biol 1994;127: 2061-2069.

29.

Ress A. , Moelling K. : Bcr interferes with β-catenin-Tcf1 interaction. FEBS Lett 2006;580:1227-1230.

30.

Haraguchi K. , Nishida A. , Ishidate T. , Akiyama T. : Activation of β-catenin-TCF-mediated transcription by non-receptor tyrosine kinase v-Src. Biochem Biophys Res Commun 2004 ;313:841-844.