Ezrin is a cytoplasmic linker molecule between plasma membrane components and the actin-containing cytoskeleton. We studied whether ezrin is associated with intercellular adhesion molecule (ICAM)-1, -2, and -3. In transfected cells, ICAM-1 and ICAM-2 colocalized with ezrin in microvillar projections, whereas an ICAM-1 construct attached to cell membrane via a glycophosphatidylinositol anchor was uniformly distributed on the cell surface. An interaction of ICAM-2 and ezrin was seen by affinity precipitation, microtiter binding assay, coimmunoprecipitation, and surface plasmon resonance methods. The calculated K D value was 3.3 ؋ 10 ؊7 M. Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ) induced an interaction of ezrin and ICAM-1 and enhanced the interaction of ezrin and ICAM-2, but ICAM-3 did not bind ezrin even in the presence of PtdIns(4,5)P 2 . PtdIns(4,5)P 2 was shown to bind to cytoplasmic tails of ICAM-1 and ICAM-2, which are the first adhesion proteins demonstrated to interact with PtdIns(4,5)P 2 . The results indicate an interaction of ezrin with ICAM-1 and ICAM-2 and suggest a regulatory role of phosphoinositide signaling pathways in regulation of ICAM-ezrin interaction.

Mutations in the NF2 tumor suppressor gene encoding merlin induce the development of tumors of the nervous system. Merlin is highly homologous to the ERM (ezrinradixin-moesin) family of membrane/cytoskeleton linker proteins. However, the mechanism for the tumor suppressing activity of merlin is not well understood. Previously, we characterized a novel role for merlin as a protein kinase A (PKA)-anchoring protein, which links merlin to the cAMP/PKA signaling pathway. In this study we show that merlin is also a target for PKAinduced phosphorylation. In vitro [␥-33 P]ATP labeling revealed that both the merlin N and C termini are phosphorylated by PKA. Furthermore, both in vitro and in vivo phosphorylation studies of the wild-type and mutated C termini demonstrated that PKA can phosphorylate merlin at serine 518, a site that is phosphorylated also by p21-activated kinases (PAKs). Merlin was phosphorylated by PKA in cells in which PAK activity was suppressed, indicating that the two kinases function independently. Both in vitro and in vivo interaction studies indicated that phosphorylation of serine 518 promotes heterodimerization between merlin and ezrin, an event suggested to convert merlin from a growthsuppressive to a growth-permissive state. This study provides further evidence on the connection between merlin and cAMP/PKA signaling and suggests a role for merlin in the cAMP/PKA transduction pathway.

Mesenchymal stem/stromal cells (MSCs) have the capacity to counteract excessive inflammatory responses. MSCs possess a range of immunomodulatory mechanisms, which can be deployed in response to signals in a particular environment and in concert with other immune cells. One immunosuppressive mechanism, not so well-known in MSCs, is mediated via adenosinergic pathway by ectonucleotidases CD73 and CD39. In this study, we demonstrate that adenosine is actively produced from adenosine 5 0 -monophosphate (AMP) by CD73 on MSCs and MSC-derived extracellular vesicles (EVs). Our results indicate that although MSCs express CD39 at low level and it colocalizes with CD73 in bulge areas of membranes, the most efficient adenosine production from adenosine 5 0 -triphosphate (ATP) requires co-operation of MSCs and activated T cells. Highly CD39 expressing activated T cells produce AMP from ATP and MSCs produce adenosine from AMP via CD73 activity. Furthermore, adenosinergic signaling plays a role in suppression of T cell proliferation in vitro. In conclusion, this study shows that adenosinergic signaling is an important immunoregulatory mechanism of MSCs, especially in situations where ATP is present in the extracellular environment, like in tissue injury. An efficient production of immunosuppressive adenosine is dependent on the concerted action of CD39-positive immune cells with CD73-positive cells such as MSCs or their EVs. STEM CELLS 2016;34:781-790 SIGNIFICANCE STATEMENTWe have studied immunomodulatory mechanism not so well known in human mesenchymal stromal cells (MSCs), namely adenosinergic signaling mediated by ectonucleotidases CD73 and CD39. We believe that adenosinergic signaling is particularly important mechanism of MSCs in tissue damage where nucleotides such as ATP are abundantly available in the extracellular environment. Human MSCs and MSC-derived extracellular vesicles, which highly express CD73, efficiently produce adenosine from AMP. However, MSCs and CD39-expressing immune cells (such as activated T cells) co-operate in the production of adenosine from ATP. In addition, MSCs can suppress T cell proliferation in an in vitro assay via adenosinergic signaling, when ATP is added to the assay. This mechanism may have been overlooked in the standard potency assays and also in vivo.

Single-chain antibodies consist of the variable, antigen-binding domains of antibodies joined to a continuous polypeptide by genetically engineered peptide linkers. We have used the flexible interdomain linker region of a fungal cellulase to link together the variable domains of an anti-2-phenyloxazolone IgG1 and show here that the resulting single-chain antibody is efficiently secreted and released to the culture medium of Escherichia coli. The yield of affinity-purified single-chain antibody is 1-2 mg/l of culture medium and its affinity and stability are comparable to those of the corresponding native IgG.