Integrins are adhesion receptors capable of transmitting intracellular signals that regulate many different cellular functions. Among integrin-mediated signals, the activation of ion channels can be included. We demonstrated that a long-lasting activation of hERG (human ether-a-go-go-related gene) potassium channels occurs in both human neuroblastoma and leukaemia cells after the activation of the β1 integrin subunit. This activation is apparently a determining factor inducing neurite extension and osteoclastic differentiation in both the cell types. More recently, we provided evidences that β1 integrins and hERG channels co-precipitate in both the cell types. Preliminary results suggest that a macromolecular signalling complex indeed occurs between integrins and the hERG1 protein and that hERG channel activity can modulate integrin downstream signalling.
Adhesive receptors of the integrin family mediate cell adhesion on the extracellular matrix. In addition to anchoring the extracellular matrix to the actin cytoskeleton, integrins trigger multiple signalling pathways that regulate cell migration, proliferation and differentiation and prevent apoptosis . The signalling properties of integrin receptors can often be traced back to their ability to form physical complexes at the cell membrane with cell-surface receptors, giving rise to signalling platforms localized at the adhesive sites . In particular, integrins are often recovered with Caveolin-1, a protein that characterizes a special plasma-membrane microdomain, i.e. caveolae . Caveolae are detergent-resistant membrane areas belonging to the so-called lipid rafts, which not only represent an alternative endocytic pathway, but also seem to act as organized transducing centres that concentrate key signalling molecules . Integrin association in lipid rafts/caveolae modulates integrin signalling, but the function of this process is not clear .
Evidences have also emerged for the idea that integrins are functionally associated with different types of ion channels [5,6]. The interaction between integrins and ion channels might have a prominent role in the regulation of different cellular activities. The largest number of studies indicates that integrins can regulate ion-channel activity. For example, integrin-dependent adhesion initiates Ca2+ influx in various types of cells: endothelial cells, fibroblasts, osteoclasts, leucocytes, hepatocytes, smooth-muscle cells and epithelial cells . In vascular-muscle cells, Ca2+ channel activation after integrin engagement leads to the regulation of cell tone and constriction . In human neutrophils, activation of the β2 subunit of the integrin receptor triggers Cl− efflux, which is necessary to regulate both spreading and respiratory burst . We also demonstrated that a long-lasting activation of hERG (human ether-a-go-go-related gene) K+ channels occurs in neuronal and haemopoietic tumour cells after integrin-mediated adhesion [7–9]. In these cell lines, hERG activation is associated with the induction of neurite extension and osteoclastic differentiation respectively.
Some of the above-mentioned integrin-mediated effects on ion channels have been shown to depend on tyrosine phosphorylation . In other cases, it is, instead, the ion-channel activation subsequent to integrin-mediated adhesion that regulates the phosphorylated state of intracellular proteins, such as FAK (focal adhesion kinase) .
K+ channels can also regulate integrin expression: in human preosteoclastic cells, hERG channel activation is apparently responsible for an up-regulation of αvβ3 expression on the plasma membrane . Kv 1.3 channels are necessary for the activation of β1 integrins and for subsequent integrin-dependent adhesion and migration in T lymphocytes .
Another important aspect of integrin–ion-channel interaction is that integrins may contribute to localization of ion channels on to the membrane. The above-mentioned functional activation of β1 integrins by Kv 1.3 channels actually relies on the physical association between the two molecules in T lymphocytes and melanoma cells. In the latter, Kv 1.3–β1 integrin interaction is promoted by cell adhesion and inhibited by channel blockers. β1 integrins also assemble with GIRK (G-protein-gated inwardly rectifying K+) channels in reconstituted systems (oocytes) and, hence, modulate channel activity. A Ca2+-activated Cl− channel, CLCA2, serves as a β4 integrin-binding partner for adhesion between endothelial cells and breast cancer cells: this interaction seems to be involved in the regulation of tumour metastasis (reviewed in ).
As stated above, our previous work in this field allowed us to demonstrate that hERG channels are functionally activated by β1 integrins in neuroblastoma and leukaemia cells [7–9]. Such a functional association might be traced back to the occurrence of a macromolecular complex between the integrin subunit and the hERG proteins. It is worth recalling here that the hERG channel is a voltage-gated K+ channel of the EAG (ether-a-go-go) family that constitutes the molecular basis for the cardiac repolarizing current known as Ikr . hERG currents can also regulate cell firing in excitable cells and contribute to the regulation of cell proliferation and invasiveness in tumour cells (reviewed in [6,13]). Functional hERG channels are tetramers and each subunit consists of a six-transmembrane protein, with both N- and C-termini located intracellularly. Neuroblastoma and leukaemia tumour cells, in which the above functional link occurs, express heterotetrameric hERG channels on the plasma membrane, composed of both full-length hERG1 and the splice variant hERG1B proteins .
We showed recently that the β1 integrin co-precipitate with hERG channels in neuroblastoma cells . To understand better this topic, we used a cellular model consisting of HEK-293 cells (human embryonic kidney 293 cells) transfected with different hERG isoforms. We therefore demonstrate that a macromolecular complex occurs only between the β1 integrin subunit and the N-terminal domain of the full-length hERG protein. In HEK-293-transfected cells, this complex localizes in caveolae/lipid rafts and behaves as a signalling complex: in fact, it can recruit the tyrosine kinase FAK as well as the small GTPase, Rac1. Similar to what was suggested in neuroblastoma cells , both FAK phosphorylation and Rac1 activity were apparently dependent on hERG current activity (A. Cherubini, G. Hofmann, S. Pillazzi, L. Guasti, O. Crociani, E. Cilia, M. Balzi, R. Wymore, S. Degani, P. Di Stefano, P. Defilippi, E. Wanke, A. Becchetti and A. Arcangeli, unpublished work).
The main message gathered from our results is that a β1–hERG complex occurs in various cell types and leads to the hERG current activation. Association between the two proteins may target hERG channels into caveolae/lipid rafts; in these sites, activated hERG channels could represent modulators of integrin downstream signalling. It is tempting to speculate that the K+ flow operated by hERG current activation could create electrostatic forces that can recruit to the plasma membrane (and, in turn, activate) a voltage-dependent signalling protein.
Signalling Outwards and Inwards: A Focus Topic at BioScience2004, held at SECC Glasgow, U.K., 18–22 July 2004. Edited by J. Challiss (Leicester, U.K.), A. Harwood (University College London, U.K.), M. Humphries (Manchester, U.K.), C. Isacke (Institute of Cancer Research, London, U.K.), R. Liddington (Burnham Institute, La Jolla, CA, U.S.A.), T. Palmer (Glasgow, U.K.), K. Siddle (Cambridge, U.K.), C. Sutherland (Dundee, U.K.), H. Wallace (Aberdeen, U.K.) and M. Welham (Bath, U.K.).
This work was supported by grants from the Telethon Fondazione Onlus (project no. GGP02208) and Ministero dell'Università e della Ricerca Scientifica e tecnologica (MURST Cofin'03) to A.A.