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Genetically determined proteolytic cleavage modulates alpha7beta1 integrin function.

ABSTRACT: The dystrophin-glycoprotein complex and the alpha7beta1 integrin are trans-sarcolemmal linkage systems that connect and transduce contractile forces between muscle fibers and the extracellular matrix. alpha7beta1 is the major laminin binding integrin in skeletal muscle. Different functional variants of this integrin are generated by alternative splicing and post-translational modifications such as glycosylation and ADP-ribosylation. Here we report a species-specific difference in alpha7 chains that results from an intra-peptide proteolytic cleavage, by a serine protease, at the 603RRQ605 site. Site-directed mutagenesis of RRQ to GRQ prevents this cleavage. This RRQ sequence in the alpha7 integrin chain is highly conserved among vertebrates but it is absent in mice. Protein structure modeling indicates this cleavage site is located in an open region between the beta-propeller and thigh domains of the alpha7 chain. Compared with the non-cleavable alpha7 chain, the cleaved form enhances cell adhesion and spreading on laminin. Cleavage of the alpha7 chain is elevated upon myogenic differentiation, and this cleavage may be mediated by urokinase-type plasminogen activator. These results suggest proteolytic cleavage is a novel mechanism that regulates alpha7 integrin functions in skeletal muscle, and that the generation of such cleavage sites is another evolutionary mechanism for expanding and modifying protein functions.

SUBMITTER: Liu J

PROVIDER: S-EPMC2602887 | biostudies-literature | 2008 Dec

REPOSITORIES: biostudies-literature

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Genetically determined proteolytic cleavage modulates alpha7beta1 integrin function.

Liu Jianming J Gurpur Praveen B PB Kaufman Stephen J SJ

The Journal of biological chemistry 20081021 51

The dystrophin-glycoprotein complex and the alpha7beta1 integrin are trans-sarcolemmal linkage systems that connect and transduce contractile forces between muscle fibers and the extracellular matrix. alpha7beta1 is the major laminin binding integrin in skeletal muscle. Different functional variants of this integrin are generated by alternative splicing and post-translational modifications such as glycosylation and ADP-ribosylation. Here we report a species-specific difference in alpha7 chains t ...[more]

PMID: 18940796

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Project description:Calreticulin presentation on the cell surface is an important hallmark of immunogenic cell death (ICD), serving as the prophagocytic signal for macrophages. Cell adhesion is a physiologically relevant stimulus previously shown to increase calreticulin interaction with ?-integrins via the juxtamembrane, cytosolic GFFKR motif. This study assessed whether integrin function can regulate surface calreticulin levels in ICD. We generated calreticulin-null T-lymphoblasts and confirmed the loss of surface calreticulin expression on cells treated with doxorubicin, an ICD inducer. Reconstituted expression with full-length calreticulin targeted to the endoplasmic reticulum (ER) successfully rescued doxorubicin-induced surface calreticulin. Reconstitution with a truncation mutant calreticulin targeted to the cytosol led to constitutively high surface calreticulin that was not further elevated by doxorubicin, suggesting calreticulin released from the stressed ER transits the cytosol before its translocation to the cell surface. When stimulated to engage integrin substrates, doxorubicin-treated wild-type T-lymphoblasts exhibited decreased surface calreticulin compared with cells under non-adherent conditions. The inhibitory effect on surface calreticulin was recapitulated for cells in suspension treated with a ?1-integrin-activating antibody, 9EG7. Similarly, cells expressing a truncated ?-integrin cytosolic tail, bearing only the juxtamembrane GFFKR calreticulin-binding motif, exhibited low surface calreticulin with doxorubicin treatment under non-adherent conditions. Using partial permeabilization techniques to distinguish between cytosolic and ER staining, we found that ICD inducers promoted the accumulation of cytosolic calreticulin with negligible change in total calreticulin, suggesting that integrin-mediated inhibition of surface calreticulin was due to reduced cytosolic to surface translocation. T-lymphoblasts co-treated with an ICD inducer and 9EG7 exhibited reduced phagocytosis by macrophages when compared with treatment with only ICD inducer. This study reveals a previously uncharacterized function of integrins as negative regulators of ICD by suppressing presentation of cell surface calreticulin.

Project description:BACKGROUND:Neuronal cell loss contributes to the pathology of acute and chronic neurodegenerative diseases, including Alzheimer's disease (AD). It remains crucial to identify molecular mechanisms sensitizing neurons to various insults and cell death. To date, the multifunctional, autophagy-related protein Beclin 1 has been shown to be both necessary and sufficient for neuronal integrity in neurodegenerative models associated with protein aggregation. Interestingly, besides its role in cellular homeostasis, Beclin 1 has also been ascribed a role in apoptosis. This makes it critical to elucidate whether Beclin 1 regulates neuronal death and survival across neurodegenerative conditions independent of protein clearance. Here, we provide experimental evidence for a direct functional link between proteolytic cleavage of Beclin 1 and apoptotic neuronal cell loss in two independent models of neurodegeneration in vivo. METHODS:Proteolytic cleavage of Beclin 1 was characterized in lysates of human AD brain samples. We developed viral tools allowing for the selective neuronal expression of the various Beclin 1 forms, including Beclin 1 cleavage products as well as a cleavage-resistant form. The effect of these Beclin 1 forms on survival and integrity of neurons was examined in models of acute and chronic neurodegeneration in vitro and in vivo. Markers of neuronal integrity, neurodegeneration and inflammation were further assessed in a Kainic acid-based mouse model of acute excitotoxic neurodegeneration and in a hAPP-transgenic mouse model of AD following perturbation of Beclin 1 in the susceptible CA1 region of the hippocampus. RESULTS:We find a significant increase in caspase-mediated Beclin 1 cleavage fragments in brain lysates of human AD patients and mimic this phenotype in vivo using both an excitotoxic and hAPP-transgenic mouse model of neurodegeneration. Surprisingly, overexpression of the C-terminal cleavage-fragment exacerbated neurodegeneration in two distinct models of degeneration. Local inhibition of caspase activity ameliorated neurodegeneration after excitotoxic insult and prevented Beclin 1 cleavage. Furthermore, overexpression of a cleavage-resistant form of Beclin 1 in hippocampal neurons conferred neuroprotection against excitotoxic and Amyloid beta-associated insults in vivo. CONCLUSIONS:Together, these findings indicate that the cleavage state of Beclin 1 determines its functional involvement in both neurodegeneration and neuroprotection. Hence, manipulating the cleavage state of Beclin 1 may represent a therapeutic strategy for preventing neuronal cell loss across multiple forms of neurodegeneration.

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Genetically determined proteolytic cleavage modulates alpha7beta1 integrin function.

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Genetically determined proteolytic cleavage modulates alpha7beta1 integrin function.

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