Interaction of Actin with Carcinoembryonic Antigen-related Cell Adhesion Molecule 1 (CEACAM1) Receptor in Liposomes Is Ca2+- and Phospholipid-dependent

Rongze Lu,Michiel J.M Niesen,Weidong Hu,Nagarajan Vaidehi,John E Shively

doi:10.1074/jbc.m111.235762

Rongze Lu, Michiel J.M Niesen + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m111.235762

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Abstract

The regulation of binding of G-actin to cytoplasmic domains of cell surface receptors is a common mechanism to control diverse biological processes. To model the regulation of G-actin binding to a cell surface receptor we used the cell-cell adhesion molecule carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1-S) in which G-actin binds to its short cytoplasmic domain (12 amino acids; Chen, C. J., Kirshner, J., Sherman, M. A., Hu, W., Nguyen, T., and Shively, J. E. (2007) J. Biol. Chem. 282, 5749-5760). A liposome model system demonstrates that G-actin binds to the cytosolic domain peptide of CEACAM1-S in the presence of negatively charged palmitoyl-oleoyl phosphatidylserine (POPS) liposomes and Ca(2+). In contrast, no binding of G-actin was observed in palmitoyl-oleoyl phosphatidylcholine (POPC) liposomes or when a key residue in the peptide, Phe-454, is replaced with Ala. Molecular Dynamics simulations on CEACAM1-S in an asymmetric phospholipid bilayer show migration of Ca(2+) ions to the lipid leaflet containing POPS and reveal two conformations for Phe-454 explaining the reversible availability of this residue for G-actin binding. NMR transverse relaxation optimized spectroscopic analysis of (13)C-labeled Phe-454 CEACAM1-S peptide in liposomes plus actin further confirmed the existence of two peptide conformers and the Ca(2+) dependence of actin binding. These findings explain how a receptor with a short cytoplasmic domain can recruit a cytosolic protein in a phospholipid and Ca(2+)-specific manner. In addition, this model system provides a powerful approach that can be applied to study other membrane protein interactions with their cytosolic targets.

Highlights

Form” of CEACAM13 stands out as a rather simple example in that it is a single-pass transmembrane protein with a cytoplasmic domain of only 12 amino acids
When a single amino acid phenylalanine 454 in the cytoplasmic domain is mutated to alanine (F454A), it no longer binds G-actin in in vitro assays, and when transfected into MCF7 cells that form a lumen with wildtype CEACAM1-S, it no longer forms a lumen in three-dimensional culture (5)
Intrigued by the ability of such a short stretch of amino acids to convey G-actin binding in response to the homotypic cell-cell interaction function of CEACAM1, we speculated that the adjacent membrane environment and Ca2ϩ signaling may play a role in regulating the binding, otherwise binding would be constitutive and irreversible

Summary

Introduction

Form” of CEACAM13 stands out as a rather simple example in that it is a single-pass transmembrane protein with a cytoplasmic domain of only 12 amino acids. We generated an in vitro model of the peptide-actin interaction in the context of negatively charged liposomes in the presence or absence of Ca2ϩ and analyzed their interactions by a combination of fluorescent bead analysis, NMR TROSY experiment, molecular dynamics (MD) simulation, and surface plasmon resonance analysis.

Results

Conclusion