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  • Original Paper
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The adaptor Grb7 is a novel calmodulin-binding protein: functional implications of the interaction of calmodulin with Grb7

Oncogene volume 24, pages 4206–4219 (2005)Cite this article

Abstract

We demonstrate using Ca2+-dependent calmodulin (CaM)-affinity chromatography and overlay with biotinylated CaM that the adaptor proteins growth factor receptor bound (Grb)7 and Grb7V (a naturally occurring variant lacking the Src homology 2 (SH2) domain) are CaM-binding proteins. Deletion of an amphiphilic basic amino-acid sequence (residues 243–256) predicted to form an α-helix located in the proximal region of its pleckstrin homology (PH) domain demonstrates the location of the CaM-binding domain. This site is identical in human and rodents Grb7, and shares great homology with similar regions of Grb10 and Grb14, and the Mig10 protein from Caenorhabditis elegans. We show that Grb7 and Grb7V are present in the cytosol and bound to membranes, while the deletion mutants (Grb7Δ and Grb7VΔ) have less capacity to be associated to membranes. Grb7Δ maintains in part the capacity to bind phosphoinositides, and CaM competes for phosphoinositide binding. Activation of ErbB2 by heregulin β1 decreases the pool of Grb7 associated to membranes. The cell-permeable CaM antagonist W7 (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide), but not the CaM-dependent protein kinase II inhibitor KN93, prevents this effect. Highly specific cell-permeable CaM inhibitory peptides decrease the association of Grb7 to membranes. This suggests that CaM regulates the intracellular mobilization of Grb7 in living cells. Direct interaction between enhanced yellow fluorescent protein (EYFP)-Grb7 and enhanced cyan fluorescent protein (ECFP)-CaM chimeras at the plasma membrane of living cells was demonstrated by fluorescence resonance energy transfer (FRET). The FRET signal dramatically decreased in cells loaded with a cell-permeable Ca2+ chelator, and was significantly attenuated when enhanced yellow fluorescent protein-Grb7 chimera (EYFP-Grb7)Δ instead of EYFP-Grb7 was used. Finally, we show that conditioned media from cells transiently transfected with Grb7Δ and Grb7VΔ lost its angiogenic activity, in contrast to those from cells transiently transfected with their wild-type counterparts.

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Abbreviations

aCtrl-P:

acylated control peptide

aMLCK-P:

acylated myosin light-chain kinase peptide

BAPTA-AM:

[1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetra(acetoxymethyl) ester]

B-CaM:

biotinylated calmodulin

CaM:

calmodulin

CaM-BD:

CaM-binding domain

CaMK-II:

CaM-dependent protein kinase II

DAPI:

4′,6-diamidino-2-phenylindole

DMEM:

Dulbecco‘s modified Eagle's medium

ECFP-CaM:

enhanced cyan fluorescent protein-calmodulin chimera

EYFP-Grb7:

enhanced yellow fluorescent protein-Grb7 chimera

EGF:

epidermal growth factor

EGFR:

EGF receptor

EGFR-GFP:

EGFR-green fluorescent protein chimera

EDTA:

ethylenediaminetetraacetic acid

EGTA:

[ethylene bis(oxyethylenenitrilo)]-tetraacetic acid

FAK:

focal adhesion kinase

FBS:

fetal bovine serum

FITC:

fluorescein isothiocyanate

FRET:

fluorescence resonance energy transfer

Grb:

growth factor receptor bound

HEPES:

N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid

HRGβ1:

heregulin β1

IPTG:

isopropyl-β-D-thiogalactopyranoside

mCtrl-P:

myristoylated control peptide

mMLCK-P:

myristoylated myosin light-chain kinase peptide

PBS:

phosphate-buffered saline

PCR:

polymerase chain reaction

PH:

pleckstrin homology

Pipes:

piperazine-N,N′-bis(ethanesulfonic acid)

PMSF:

phenylmethylsulfonyl fluoride

P-Tyr:

phosphotyrosine

PVDF:

polyvinylidene fluoride

SH2:

Src homology 2

W7:

N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide

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Acknowledgements

We thank Professors Shinji Tanaka for Grb7 and Grb7V vectors, Nobuhiro Hayashi for recombinant calmodulin, Atushi Miyawaki for the ECFP-CaM vector, Carlos Enrich for PAE/EGFR-GFP cells, Ms M Dolores Morales-García for assistance with the FRET experiments, Ms Amparo Jiménez and Ms Marguerite Ball for technical assistance, and Dr Danica Stanimirovic for resources and facilities to perform the angiogenesis experiments. This work was financed by grants (to AV) from the Comisión Interministerial de Ciencia y Tecnología (SAF2002-03258) and the Instituto Carlos III, Fondo de Investigaciones Sanitarias (RTICCC C03/10). HL was supported by the Agencia Española de Cooperación Internacional (2002CN0013), and AFdC by the Fundación Carolina.

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  1. Hongbing Li and Juan Sánchez-Torres: These authors contributed equally to this work

Authors and Affiliations

  1. Instituto de Investigaciones Biomédicas, Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid, c/Arturo Duperier 4, Madrid, E-28029, Spain

    Hongbing Li, Juan Sánchez-Torres, Alan F del Carpio, Aitor Nogales-González, Patricia Molina-Ortiz & Antonio Villalobo

  2. Institute for Biological Sciences, National Research Council, 1500 Montreal Road, Ottawa, Ontario, Canada, K1A 0R6

    María J Moreno

  3. Department of Basic Medical Science, Pharmacology and Clinical Pharmacology Section, St George Hospital Medical School, Cranmer Terrace, London, SW17 0RE, UK

    Katalin Török

  4. Science and Research Center, Shijiazhuang Medical School, 450Zhongshan Xi Road, Shijiazhuang, Hebei, 05008, People's Republic of China

    Hongbing Li

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Li, H., Sánchez-Torres, J., del Carpio, A. et al. The adaptor Grb7 is a novel calmodulin-binding protein: functional implications of the interaction of calmodulin with Grb7. Oncogene 24, 4206–4219 (2005). https://doi.org/10.1038/sj.onc.1208591

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