Expression cloning of signaling proteins regulated by cell adhesion.
Many proteins involved in cell-cell and cell-matrix adhesion are regulated by signal transduction pathways and can activate signal transduction on ligation. Adhesion-related signal transduction is important throughout development, hemostasis, immunity, and in diseases such as cancer. Therefore, the identification of the various signaling pathways that are involved is crucial. Expression cloning is an unbiased way to isolate proteins with specific biological functions. This methodology has been adapted for the identification of proteins involved in cell signaling pathways that are mediated by cell-cell and cell-matrix interactions. We have successfully developed and used a novel expression cloning strategy to isolate the integrin-regulated apoptosis signaling protein BIT-1. This screen was based on previous observations that integrin-mediated adhesion upregulates the anti-apoptotic protein bcl-2. Our strategy described in this chapter uses flow cytometry and a reporter construct in which the bcl-2 promoter is linked to enhanced green fluorescence protein. The advantage of using flow cytometry in expression cloning is that it increases the sensitivity of the screen by enabling us to examine function quantitatively at the level of a single cell millions of times in one experiment. The following protocol provides a detailed method for the isolation of proteins that are regulated by cell adhesion.
Matter
Michelle L
ML
John A. Burns Medical School, University of Hawaii at Manoa, Honolulu, HI, USA.
Ramos
Joe W
JW
D000818:Animals; D016466:CHO Cells; D019556:COS Cells; D002448:Cell Adhesion / Q000235:genetics; D015815:Cell Adhesion Molecules / Q000235:genetics / Q000378:metabolism; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular / Q000379:methods; D006224:Cricetinae; D003412:Cricetulus; D005109:Extracellular Matrix / Q000235:genetics / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods; D020869:Gene Expression Profiling / Q000379:methods; D015398:Signal Transduction / Q000235:genetics*; D015854:Up-Regulation / Q000235:genetics*
D015815:Cell Adhesion Molecules