The neurofibromatosis-2 (NF2) gene encodes merlin, an ezrin-radixin-moesin-(ERM)-related protein that functions as a tumor suppressor. We found that merlin mediates contact inhibition of growth through signals from the extracellular matrix. At high cell density, merlin becomes hypo-phosphorylated and inhibits cell growth in response to hyaluronate (HA), a mucopolysaccharide that surrounds cells. Merlin's growth-inhibitory activity depends on specific interaction with the cytoplasmic tail of CD44, a transmembrane HA receptor. At low cell density, merlin is phosphorylated, growth permissive, and exists in a complex with ezrin, moesin, and CD44. These data indicate that merlin and CD44 form a molecular switch that specifies cell growth arrest or proliferation.

CD44 is a widely distributed type I transmembrane glycoprotein and functions as the major hyaluronan receptor on most cell types. Although alternative splicing can produce a large number of different isoforms, they all retain the hyaluronan-binding Link-homology region and a common transmembrane and cytoplasmic domain, which are highly conserved between species. The past decade has seen an extensive investigation of this receptor owing to its importance in mediating cell-cell and cell-matrix interactions in both normal and disease states. Although roles for alternative splicing and variable glycosylation in determining ligand-binding interactions are now well established, the mechanisms by which CD44 integrates structural and signalling events to elicit cellular responses have been less well understood. However, there is now increasing evidence that CD44 is assembled in a regulated manner into membrane-cytoskeletal junctional complexes and, through both direct and indirect interactions, serves to focus downstream signal transduction events.

The dynamic assembly and disassembly of membrane cytoskeleton junctional complexes is critical in cell migration. Here we describe a novel phosphorylation mechanism that regulates the hyaluronan receptor CD44. In resting cells, CD44 is constitutively phosphorylated at a single serine residue, Ser325. After protein kinase C is activated, a switch in phosphorylation results in CD44 being phosphorylated solely at an alternative residue, Ser291. Using fluorescence resonance energy transfer (FRET) monitored by fluorescence lifetime imaging microscopy (FLIM) and chemotaxis assays we show that phosphorylation of Ser291 modulates the interaction between CD44 and the cytoskeletal linker protein ezrin in vivo, and that this phosphorylation is critical for CD44-dependent directional cell motility.

ERM (ezrin, radixin and moesin) proteins function as linkers between the actin cytoskeleton and the plasma membrane. In addition to this structural role, these proteins are highly regulatable making them ideal candidates to mediate important physiological events such as adhesion and membrane morphology and to control formation and breakdown of membrane-cytoskeletal junctions. Recently, a direct interaction in vitro has been demonstrated between ERM proteins and the hyaluronan receptor, CD44. We have mapped the ezrin-binding site to two clusters of basic amino acids in a membrane-proximal 9 amino-acid region within the CD44 cytoplasmic domain. To investigate the functional importance of this interaction in vivo, we created a number of mutations within full-length CD44 and expressed these mutants in human melanoma cells. We demonstrate here that mutations within the ezrin-binding site do not disrupt the plasma membrane localization of CD44 and, in addition, that this region is not required to mediate efficient hyaluronan binding. These studies suggest that ERM proteins mediate the outside-in, rather than inside-out, signalling of adhesion receptors.

Human interfollicular epidermis is renewed by stem cells that are clustered in the basal layer in a patterned, non-random distribution. Stem cells can be distinguished from other keratinocytes by high expression of β1 integrins and lack of expression of terminal differentiation markers; they divide infrequently in vivo but form actively growing colonies in culture. In a search for additional stem cell markers, we observed heterogeneous epidermal expression of melanoma chondroitin sulphate proteoglycan (MCSP). MCSP was expressed by those keratinocytes with the highest β1 integrin levels. In interfollicular epidermis, expression was confined to non-cycling cells and,in culture, to self-renewing clones. However, fluorescence-activated cell sorting on the basis of MCSP and β1 integrin expression gave no more enrichment for clonogenic keratinocytes than sorting for β1 integrins alone. To interfere with endogenous MCSP, we retrovirally infected keratinocytes with a chimera of the CD8 extracellular domain and the MCSP cytoplasmic domain. CD8/MCSP did not affect keratinocyte proliferation or differentiation but the cohesiveness of keratinocytes in isolated clones or reconstituted epidermal sheets was greatly reduced. CD8/MCSP caused stem cell progeny to scatter without differentiating. CD8/MCSP did not alter keratinocyte motility but disturbed cadherin-mediated cell-cell adhesion and the cortical actin cytoskeleton, effects that could be mimicked by inhibiting Rho. We conclude that MCSP is a novel marker for epidermal stem cells that contributes to their patterned distribution by promoting stem cell clustering.

interaction dependent on the Delta1 PDZ-binding domain. Syntenin, like Delta1, is upregulated in the stem cell clusters of human interfollicular epidermis. Knockdown of syntenin in cells overexpressing full-length Delta1 had the same effects on Notch signalling, epidermal differentiation and adhesion as overexpressing Delta1 with a mutated PDZ-binding domain. Syntenin has previously been reported to regulate membrane traffic, and mutation of the Delta1 PDZ-binding domain or knockdown of syntenin led to rapid internalisation of Delta1. We propose that syntenin binding to Delta1 plays a dual role in promoting intercellular adhesion and regulating Notch signalling.

Targeted delivery of chemotherapeutics aims to increase efficacy and lower toxicity by concentrating drugs at the site-of-action, a method embodied by the seven current FDA-approved antibody–drug conjugates (ADC). However, a variety of pharmacokinetic challenges result in relatively narrow therapeutic windows for these agents, hampering the development of new drugs. Here, we use a series of prostate-specific membrane antigen–binding single-domain (Humabody) ADC constructs to demonstrate that tissue penetration of protein–drug conjugates plays a major role in therapeutic efficacy. Counterintuitively, a construct with lower in vitro potency resulted in higher in vivo efficacy than other protein–drug conjugates. Biodistribution data, tumor histology images, spheroid experiments, in vivo single-cell measurements, and computational results demonstrate that a smaller size and slower internalization rate enabled higher tissue penetration and more cell killing. The results also illustrate the benefits of linking an albumin-binding domain to the single-domain ADCs. A construct lacking an albumin-binding domain was rapidly cleared, leading to lower tumor uptake (%ID/g) and decreased in vivo efficacy. In conclusion, these results provide evidence that reaching the maximum number of cells with a lethal payload dose correlates more strongly with in vivo efficacy than total tumor uptake or in vitro potency alone for these protein–drug conjugates. Computational modeling and protein engineering can be used to custom design an optimal framework for controlling internalization, clearance, and tissue penetration to maximize cell killing. Significance: A mechanistic study of protein–drug conjugates demonstrates that a lower potency compound is more effective in vivo than other agents with equal tumor uptake due to improved tissue penetration and cellular distribution.

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