The Leucine-rich Repeat Protein LRIG1 Is a Negative Regulator of ErbB Family Receptor Tyrosine Kinases

Melanie B Laederich,Melanie Funes-Duran,Lily Yen,Ellen Ingalla,Xiuli Wu,Kermit L Carraway,Colleen Sweeney

doi:10.1074/jbc.m409703200

Melanie B Laederich, Melanie Funes-Duran + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m409703200

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Abstract

The molecular mechanisms by which mammalian receptor tyrosine kinases are negatively regulated remain largely unexplored. Previous genetic and biochemical studies indicate that Kekkon-1, a transmembrane protein containing leucine-rich repeats and an immunoglobulin-like domain in its extracellular region, acts as a feedback negative regulator of epidermal growth factor (EGF) receptor signaling in Drosophila melanogaster development. Here we tested whether the related human LRIG1 (also called Lig-1) protein can act as a negative regulator of EGF receptor and its relatives, ErbB2, ErbB3, and ErbB4. We observed that in co-transfected 293T cells, LRIG1 forms a complex with each of the ErbB receptors independent of growth factor binding. We further observed that co-expression of LRIG1 with EGF receptor suppresses cellular receptor levels, shortens receptor half-life, and enhances ligand-stimulated receptor ubiquitination. Finally, we observed that co-expression of LRIG1 suppresses EGF-stimulated transformation of NIH3T3 fibroblasts and that the inducible expression of LRIG1 in PC3 prostate tumor cells suppresses EGF- and neuregulin-1-stimulated cell cycle progression. Our observations indicate that LRIG1 is a negative regulator of the ErbB family of receptor tyrosine kinases and suggest that LRIG1-mediated receptor ubiquitination and degradation may contribute to the suppression of ErbB receptor function.

Highlights

The four members of the ErbB family of receptor tyrosine kinases (epidermal growth factor (EGF)1 receptor, ErbB2, ErbB3, and ErbB4) play key roles in mediating the development of a variety of tissues, and the aberrant activation of these receptors contributes to the growth and progression of numerous tumor types [1, 2]
Our observations indicate that LRIG1 is a negative regulator of the ErbB family of receptor tyrosine kinases and suggest that LRIG1-mediated receptor ubiquitination and degradation may contribute to the suppression of ErbB receptor function
To determine whether LRIG1 might influence the properties of human ErbB receptors, we first examined the association of the proteins by co-immunoprecipitation

Summary

MATERIALS AND METHODS

Materials and Cell Culture—EGF was purchased from Upstate Biotechnology, and glutathione S-transferase-fused NRG1␤ EGF-like domain was produced in bacteria and purified by glutathione affinity. Co-immunoprecipitation and Immunoblotting—293T cells in 100-mm dishes were transfected with each ErbB receptor without or with LRIG1 using FuGENE 6 according to procedures recommended by the manufacturer. Cells were lysed in co-immunoprecipitation buffer [19], and cleared lysates were immunoprecipitated with 1.5 ␮g of anti-Myc or anti-receptor antibodies. Biotinylation Experiments—293T cells in 100-mm dishes were transfected with cDNAs encoding ErbB receptors or LRIG1, and proteins were allowed to express for 30 h. Cells were rinsed twice with phosphate-buffered saline, lysed in co-immunoprecipitation buffer, and precipitated with appropriate anti-Myc or antireceptor antibodies. Ubiquitinated receptors in PC3-LRIG1 cells were detected by blotting with anti-ubiquitin monoclonal antibody (Covance). Cells were treated with 10% serum, 50 nM NRG1␤, or 33 nM EGF for various times, collected, and fixed in 70% ethanol/phosphate-buffered saline for a minimum of 2 h at 4 °C. Analysis was based on 20,000 cells and was linearly scaled

RESULTS AND DISCUSSION

It is noteworthy that the physical mechanisms by which

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