Charo Gonzalez-Agosti

NHE-RF, a Regulatory Cofactor for Na+-H+Exchange, Is a Common Interactor for Merlin and ERM (MERM) Proteins

We have identified the human homologue of a regulatory cofactor of Na+-H+ exchanger (NHE-RF) as a novel interactor for merlin, the neurofibromatosis 2 tumor suppressor protein. NHE-RF mediates protein kinase A regulation of Na+-H+ exchanger NHE3 to which it is thought to bind via one of its two PDZ domains. The carboxyl-terminal region of NHE-RF, downstream of the PDZ domains, interacts with the amino-terminal protein 4.1 domain-containing segment of merlin in yeast two-hybrid assays. This interaction also occurs in affinity binding assays with full-length NHE-RF expressed in COS-7 cells. NHE-RF binds to the related ERM proteins, moesin and radixin. We have localized human NHE-RF to actin-rich structures such as membrane ruffles, microvilli, and filopodia in HeLa and COS-7 cells, where it co-localizes with merlin and moesin. These findings suggest that hNHE-RF and its binding partners may participate in a larger complex (one component of which might be a Na+-H+exchanger) that could be crucial for the actin filament assembly activated by the ERM proteins and for the tumor suppressor function of merlin.

Interdomain interaction of merlin isoforms and its influence on intermolecular binding to NHE-RF.

Merlin, the neurofibromatosis 2 tumor suppressor protein, has two major isoforms with alternate C termini and is related to the ERM (ezrin, radixin, moesin) proteins. Regulation of the ERMs involves intramolecular and/or intermolecular head-to-tail associations between family members. We have determined whether merlin undergoes similar interactions, and our findings indicate that the C terminus of merlin isoform 1 is able to associate with its N-terminal domain in a head-to-tail fashion. However, the C terminus of isoform 2 lacks this property. Similarly, the N terminus of merlin can also associate with C terminus of moesin. We have also explored the effect of merlin self-association on binding to the regulatory cofactor of Na+-H+exchanger (NHE-RF), an interacting protein for merlin and the ERMs. Merlin isoform 2 captures more NHE-RF than merlin isoform 1 in affinity binding assays, suggesting that in full-length merlin isoform 1, the NHE-RF binding site is masked because of the self-interactions of merlin. Treatment with a phospholipid known to decrease self-association of ERMs enhances the binding of merlin isoform 1 to NHE-RF. Thus, although isoform 1 resembles the ERM proteins, which transition between inactive (closed) and active (open) states, isoform 2 is distinct, existing only in the active (open) state and presumably constitutively more available for interaction with other protein partners.

Expression of NF2-encoded Merlin and Related ERM Family Proteins in the Human Central Nervous System

Germline mutations of the neurofibromatosis 2 (NF2) gene are associated with an increased incidence of gliomas and glial harmartomas, suggesting a role for the NF2-encoded protein, merlin, in glial growth control. Using monoclonal and polyclonal anti-merlin antibodies for Western blotting and immunohistochemistry, we evaluated the cellular pattern of merlin expression in the normal human central nervous system (CNS), reactive gliosis; and NF2-associated glial hamartomas. In the normal CNS, merlin is widely expressed in coarse cytoplasmic granules in both glia and neurons, with less pronounced expression in other cells. Merlin is also expressed in reactive astrocytes and in the astrocytes of NF2-associated glial hamartomas. In reactive astrocytes, however, merlin is also present at the cell membrane and in cellular processes, suggesting redistribution of the protein in activated cells. Merlin is structurally related to ezrin, radixin and moesin, which are also expressed in the CNS, as demonstrated by Western blotting. The pattern of merlin expression, however, is distinct from that of ezrin, which has been previously described, and that of moesin, in which immunohistochemistry with an anti-moesin antibody showed expression in endothelial cells, glia and neurons in a membranous or diffuse cytoplasmic pattern. These findings imply that merlin has widespread and specific functions in the human central nervous system.

Response of radixin to perturbations of growth cone morphology and motility in chick sympathetic neurons in vitro

The ERM protein--ezrin, radixin, moesin--localize to a variety of cortical structures, where they may participate in connecting the cytoskeleton to components of the plasma membrane. Antibodies that recognize the ERM proteins specifically stain growth cones of various neurons [Goslin et al., 1989: J. Cell Biol. 109:1621-1631; Birgbauer et al., 1991: J. Neurosci. Res. 30:232-241]. To probe the function of ERM proteins in growth cones, we studied the consequences of perturbing growth cone morphology and motility of cultured chick sympathetic neurons. We demonstrate that radixin is present in these growth cones. Withdrawal of nerve growth factor (NGF) induces rapid collapse of the growth cones; concomitantly, radixin staining in these growth cones are greatly diminished. Upon readdition of NGF, rapid growth cone formation is accompanied by relocalization of radixin. Induction of growth cone collapse by either growth cone-growth cone contact or exposure to brain membrane extract results in a similar diminution of radixin staining. We induced a more subtle change in the organization of the growth cones by subjecting them to an electric field. These growth cones rapidly orient toward the cathode. We show that the radixin staining of the growth cones is also asymmetrically localized toward the leading edges in the new direction of growth. The results suggest that the localization of radixin may be essential for the normal expression of growth cone morphology and function.