Children's National Health System Posters
Cytoskeletal protein radixin activates integrin aIIb ß3 by binding to its cytoplasmic tail
Document Type
Poster
Abstract Category
Basic Biomedical Sciences
Keywords
Cell aggregation; Integrin; The band 4.1, ezrin,radixin,Cytoskeleton
Publication Date
Spring 2019
Abstract
Background: Recognition of the integrin cytoplasmic tail by talin, via its phosphotyrosine binding (PTB) domain within its FERM-homologous head region, directly activates the integrin receptor. The talin is the only known PTB-domain-containing protein that can promote integrin activation. The F3 subdomain of talin is homologous to the PTB domain, a structure that exists in many cytoskeletal and signaling proteins, and represents the principal binding site within talin for the integrin cytoplasmic domains. Indeed, the PTB/F3 domain has equivalent integrin activating activity to the entire talin protein. Objective: To see if other FERM-domain-containing proteins are also capable of activating integrins, we previously reported interactions between radixin and integrin αM β2. Here we studied talin PTB/F3 and radixin activate integrin αIIb β3. Methods/Design: Expression of recombinant radixin in E.coli. To express the radixin N-ERMAD (1-298) and C-ERMAD (281-584) both as glutathione S-transferase (GST) fusion proteins in E.coli, we amplified their corresponding cDNAs by RT-PCR from total RNA prepared from murine macrophages. Establishment of Chinese hamster cells (CHO) expressing both integrin αIIb β3 and radixin. To express both αIIb β3 and radixin in CHO cells, cDNAs encoding N-ERMAD and C-ERMAD were inserted individually into the expression vector MGIN. Cell aggregation assays and FACS analysis were performed. Results/Discussion: The ability of the radixin FERM domain to interact with the αIIb β3 receptor is demonstrated by pull-down assays using total cell lysates. Most importantly, we found that expression of radixin in αIIb β3 –bearing cells significantly enhances its aggregation activity.
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Open Access
1
Cytoskeletal protein radixin activates integrin aIIb ß3 by binding to its cytoplasmic tail
Background: Recognition of the integrin cytoplasmic tail by talin, via its phosphotyrosine binding (PTB) domain within its FERM-homologous head region, directly activates the integrin receptor. The talin is the only known PTB-domain-containing protein that can promote integrin activation. The F3 subdomain of talin is homologous to the PTB domain, a structure that exists in many cytoskeletal and signaling proteins, and represents the principal binding site within talin for the integrin cytoplasmic domains. Indeed, the PTB/F3 domain has equivalent integrin activating activity to the entire talin protein. Objective: To see if other FERM-domain-containing proteins are also capable of activating integrins, we previously reported interactions between radixin and integrin αM β2. Here we studied talin PTB/F3 and radixin activate integrin αIIb β3. Methods/Design: Expression of recombinant radixin in E.coli. To express the radixin N-ERMAD (1-298) and C-ERMAD (281-584) both as glutathione S-transferase (GST) fusion proteins in E.coli, we amplified their corresponding cDNAs by RT-PCR from total RNA prepared from murine macrophages. Establishment of Chinese hamster cells (CHO) expressing both integrin αIIb β3 and radixin. To express both αIIb β3 and radixin in CHO cells, cDNAs encoding N-ERMAD and C-ERMAD were inserted individually into the expression vector MGIN. Cell aggregation assays and FACS analysis were performed. Results/Discussion: The ability of the radixin FERM domain to interact with the αIIb β3 receptor is demonstrated by pull-down assays using total cell lysates. Most importantly, we found that expression of radixin in αIIb β3 –bearing cells significantly enhances its aggregation activity.
Comments
Presented at Research Days 2019.