Distinct Regions of the Cadherin Cytoplasmic Domain Are Essential for Functional Interaction with Gα12 and β-Catenin

Daniel D. Kaplan,Thomas E. Meigs,Patrick J. Casey

doi:10.1074/jbc.m106121200

Abstract

Heterotrimeric G proteins of the G(12) subfamily mediate cellular signals leading to events such as cytoskeletal rearrangements, cell proliferation, and oncogenic transformation. Several recent studies have revealed direct effector proteins through which G(12) subfamily members may transmit signals leading to various cellular responses. Our laboratory recently demonstrated that Galpha(12) and Galpha(13) specifically interact with the cytoplasmic domains of several members of the cadherin family of cell adhesion molecules (Meigs, T. E., Fields, T. A., McKee, D. D., and Casey, P. J. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 519-524). This interaction causes beta-catenin to release from cadherin and relocalize to the cytoplasm and nucleus, where it participates in transcriptional activation. Here we report that two distinct regions of the epithelial cadherin (E-cadherin) tail are required for interaction with beta-catenin and Galpha(12), respectively. Deletion of an acidic, 19-amino acid region of E-cadherin abolishes its ability to bind beta-catenin in vitro, to inhibit beta-catenin-mediated transactivation, or to stabilize beta-catenin; causes subcellular mislocalization of beta-catenin; and disrupts cadherin-mediated cell adhesion. On the other hand, deletion of a distinct 11-amino acid region of E-cadherin dramatically attenuates interaction with Galpha(12); furthermore, Galpha(12) is ineffective in stimulating beta-catenin release from an E-cadherin cytoplasmic domain lacking this putative Galpha(12)-binding region. These findings indicate that Galpha(12) and beta-catenin do not compete for the same binding site on cadherin and provide molecular targets for selectively disrupting the interaction of these proteins with cadherin.

Highlights

Heterotrimeric guanine nucleotide-binding proteins (G proteins)1 regulate cellular physiology by transducing extracellular signals to intracellular effector molecules [1]
Identification of Regions of the Cadherin Cytoplasmic Domain That Interact with G␣12 and ␤-Catenin—A series of eight deletion mutants within the E-cadherin cytoplasmic domain, with each deleted region being defined by the primary sequence as proline-rich, serine-rich, acidic, basic, or mixed-charged (Fig. 1), were constructed as fusions to glutathione S-transferase (GST)
The ability of each deletion mutant to bind at least one of the two proteins (G␣12 or ␤-catenin) diminishes the likelihood that these mutations produce misfolded proteins with a global loss of protein binding capacity. These data demonstrate that distinct regions of the cadherin cytoplasmic domain are required for interaction with G␣12 and ␤-catenin

Summary

Introduction

Heterotrimeric guanine nucleotide-binding proteins (G proteins)1 regulate cellular physiology by transducing extracellular signals to intracellular effector molecules [1]. Deletion of a distinct 11-amino acid region of E-cadherin dramatically attenuates interaction with G␣12; G␣12 is ineffective in stimulating ␤-catenin release from an E-cadherin cytoplasmic domain lacking this putative G␣12-binding region. This interaction was shown to cause ␤-catenin, a multifunctional protein involved in both cell adhesion and transcriptional activation [19], to release from its cadherin-bound state and relocalize to the cytoplasm and nucleus.

Results

Conclusion