Actin-binding Protein α-Actinin-1 Interacts with the Metabotropic Glutamate Receptor Type 5b and Modulates the Cell Surface Expression and Function of the Receptor

Nuria Cabello,Rosaria Remelli,Laia Canela,Ana Soriguera,Josefa Mallol,Enric I Canela,Melanie J Robbins,Carme Lluis,Rafael Franco,R A Jeffrey Mcilhinney,Francisco Ciruela

doi:10.1074/jbc.m608880200

Nuria Cabello, Rosaria Remelli + Show 9 more

Open Access

https://doi.org/10.1074/jbc.m608880200

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Abstract

Receptors for neurotransmitters require scaffolding proteins for membrane microdomain targeting and for regulating receptor function. Using a yeast two-hybrid screen, alpha-actinin-1, a major F-actin cross-linking protein, was identified as a binding partner for the C-terminal domain of metabotropic glutamate receptor type 5b (mGlu(5b) receptor). Co-expression, co-immunoprecipitation, and pull-down experiments showed a close and specific interaction between mGlu(5b) receptor and alpha-actinin-1 in both transfected HEK-293 cells and rat striatum. The interaction of alpha-actinin-1 with mGlu(5b) receptor modulated the cell surface expression of the receptor. This was dependent on the binding of alpha-actinin-1 to the actin cytoskeleton. In addition, the alpha-actinin-1/mGlu(5b) receptor interaction regulated receptor-mediated activation of the mitogen-activated protein kinase pathway. Together, these findings indicate that there is an alpha-actinin-1-dependent mGlu(5b) receptor association with the actin cytoskeleton modulating receptor cell surface expression and functioning.

Highlights

The actin-based cytoskeleton is connected to the plasma membrane via a lattice-like network of actin-binding proteins that form the membrane skeleton or membrane-associated cytoskeleton [6]
All of them share a general structure, which can be divided into three functionally distinct domains: the N terminus containing two calponin homology domains that bind to actin filaments [9], a central region composed of four spectrin-like motifs [10], which acts as a switchboard for interactions with multiple proteins, and the C terminus, which contains EF-hand domains responsible for Ca2ϩ binding [11] and terminates in a PDZ domain-binding sequence, ESDL [12]
Yeast Two-hybrid Screening—To identify intracellular proteins interacting with the mGlu5b receptor, a region containing 178 amino acids of the C-terminal tail of the receptor were fused in-frame with LexA in the pHybLexA/Zeo vector (LmGlu5b, Fig. 1A) and used to screen a mouse brain cDNA library using the yeast two-hybrid system

Summary

Introduction

The actin-based cytoskeleton is connected to the plasma membrane via a lattice-like network of actin-binding proteins that form the membrane skeleton or membrane-associated cytoskeleton [6]. This interaction might have relevant physiological consequences, because we demonstrate, in the present work, that ␣-actinin-1 controls the cell surface expression and functioning of mGlu5b receptor.

Results

Conclusion