CNR - Institute of Neuroscience CNR
Institute of Neuroscience
 

Project

Molecular mechanisms of cell polarity

The establishment and maintenance of cell polarity within various organs are crucial for the development of all eukaryotes. The products of many genes that function coordinately to establish cell polarity are multidomain scaffold proteins that often interact each other to form large protein complexes to mediate assembly of cell junctions, organization of signal transduction complexes, regulation of cytoskeletal dynamics, and asymmetric trafficking of cell fate determinants. Many details about the function of these complexes in mammalian epithelia remain to be clarified, and our work is focused on complexes containing Lin7.

Assembly of polarity protein complexes

Lin7 was first identified in C. elegans where it forms a heterotrimeric complex with Lin2 and Lin10 that is crucial for the correct localization of the worm epidermal growth factor receptor, Let-23, to the basolateral surface of the body wall epithelium. The Lin7 mammalian homologues (also called Velis or MALS) are small proteins composed of two interaction modules: the NH2-terminal L27 domain for association to Lin2-related proteins associated to Lin7 (Pals proteins) and a COOH-terminal PDZ (domain that allow the direct interaction with proteins with a canonical type 1 PDZ interaction motif in their C-terminal end). Lin7 colocalizes with markers of adherens (AJ) and tight (TJ) junctions, where, through its interactions with structural and scaffold proteins, it may form multiprotein complexes. Indeed, the single L27 domain may interact with the first (L27N) of Pals proteins localized at AJ and TJ, and the second L27 domain of Pals1 (L27C) is known to bind Patj to form the PALS1-PATJ-CRB3 ternary complex involved in tight junction assembly in epithelial cells. We have identified as partners of the PDZ domain of LIN7 at AJs and TJs respectively β-catenin (Perego et al, EMBO J. 2000) and the insuline receptor substrate (IRS) p53 (Massari et al., Traffic 2009). Thus, Lin7 may participate in the assembly of multiprotein complexes both at the AJs and TJs, and interest of the laboratory is to identify the role of LIN7 in cell polarity.

Role of Lin7 domains in establishment and maintenance of polarized surfaces in epithelia and neurons

 

We have examined the role of the L27 [(LIN2-LIN7) domain] and PDZ domain (domain previously found in PSD95-DlgA-ZO-1) for protein-protein interaction of the scaffold protein LIN7 in tight junction (TJ) assembly in Madin-Darby canine kidney (MDCK) cells and found that the stable expression of a LIN7 mutant lacking the L27 domain acts as a dominant interfering protein by inhibiting TJ localization of endogenous LIN7.The loss of LIN7 did not alter the localization of the PALS1 (protein associated with LIN7) partner of the L27 domain but prevented TJ localization of the insulin receptor substrate p53 (IRSp53), a partner of the PDZ domain of LIN7. Cell lines with decreased localization of LIN7 and IRSp53 to TJs showed defects during assembly of TJs and cyst polarization when cultured in 3-D collagen system, and failed to activate Rac1, a member of the Rho guanosine triphosphatases family crucially involved in actin organization and orientation of apicobasal polarity.

Thus, LIN7-IRSp53 association plays a role during assembly of functional TJs and surface polarization in epithelial cells. Based on these results we propose a working model (Fig.1) for LIN7-IRSp53 functions in theregulation of TJ assembly in MDCK cellsLin7-IRSp53. Moreover, our future goals are focused on the role of Lin7-IRSp53 association during differentiation of neuronal processes.

Role of protein-protein interactions mediated by LIN7 in surface localization of the epithelial GABA transporter

 

In order to exert their vectorial functions, polarized cells require the selective localization of distinct populations of ion channels and carriers on their site of functional residence. We have shown that the lateral junctional localization of the epithelial GABA transporter BGT1 in the polarized epithelial MDCK cell model depends on the association of the transporter with the PDZ domain of Lin7. The removal of the BGT1 PDZ target motif prevents the association to LIN7 and causes the transporter internalization and accumulation in a recycling compartment (Perego et al, EMBO J, 1999). Moreover, we have also demonstrated that the association of BGT1 with LIN7 is also prevented by phosphorylation events on a threonine residue in the BGT1 PDZ target motif mediated by protein kinase C (PKC) (Massari et al, J Biol Chem, 2005), thus suggesting a mechanism based on phosphorylation events that regulates the assembly of protein complexes at the lateral junctional surface (Fig. 2)

Publications

  • Padovano V, Massari S, Mazzucchelli S, Pietrini G (2009) PKC induces internalization and retention of the EAAC1 glutamate transporter in recycling endosomes of MDCK cells. Am. J. Physiol., Cell Physiol. 297:C835-44.
  • Massari S, Perego C, Padovano V, D'Amico A, Raimondi A, Francolini M, Pietrini G (2009) LIN7 mediates the recruitment of IRSp53 to tight junctions. Traffic 10:246-57.
  • Massari S, Vanoni C, Longhi R, Rosa P, Pietrini G (2005) Protein kinase C-mediated phosphorylation of the BGT1 epithelial gamma-aminobutyric acid transporter regulates its association with LIN7 PDZ proteins: a post-translational mechanism regulating transporter surface density. J. Biol. Chem. 280:7388-97.
  • Perego C, Vanoni C, Massari S, Longhi R, Pietrini G (2000) Mammalian LIN-7 PDZ proteins associate with beta-catenin at the cell-cell junctions of epithelia and neurons. EMBO J. 19:3978-89.
  • Perego C, Vanoni C, Villa A, Longhi R, Kaech SM, Fröhli E, Hajnal A, Kim SK, Pietrini G (1999) PDZ-mediated interactions retain the epithelial GABA transporter on the basolateral surface of polarized epithelial cells. EMBO J. 18:2384-93.

Grants

Italian Ministry of University and Research, PRIN

Collaborations

  • A. Disanza, G. Scita, IFOM, Milan, Italy.

 

PI photo

Grazia Pietrini

Contact information

email  E-mail

email  +39 02 50317094

Participating staff
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