Abstract
ADP-ribosylation actor 6 (ARF6) regulates the endocytosis and recycling of a variety of proteins and also promotes peripheral actin rearrangements and cell motility. ARF6 is activated by a large number of guanine nucleotide exchange factors, which likely regulate ARF6 at different locations and during different processes. In this study we investigate the roles of the cytohesin ADP-ribosylation factor (ARF)-guanine nucleotide exchange factors during the recycling of integrin beta1. Intriguingly, we find that knockdown and overexpression of ARNO/cytohesin 2 and GRP1/cytohesin 3 have opposing effects on cell adhesion and spreading on fibronectin and on cell migration. We find that ARNO/cytohesin 2 is required for integrin beta1 recycling, whereas GRP1/cytohesin 3 is dispensable for this process. This is the first demonstration of unique roles for these proteins.
Highlights
ARF6 regulates the endocytosis of 1 integrin and a number of other membrane proteins
Cytohesins Are Critical for Cell Adhesion to Fibronectin—Integrin 1 is a component of the major fibronectin receptors
We investigated which cytohesin member is the major guanine nucleotide exchange factors (GEFs) acting in this process
Summary
ARF6 regulates the endocytosis of 1 integrin and a number of other membrane proteins. We transfected Myc-tagged ARNO or FLAG-tagged GRP1 into HeLa cells and expected that overexpression of the relevant cytohesin should enhance the adhesion in the same assay. We confirmed the differential effects of ARNO and GRP1 on cell adhesion by reducing the expression of each GEF using siRNAs. Transfection of HeLa, MCF-7, or MDCK cells with siRNAs targeting ARNO or GRP1 reduces the expression level of mRNA for these proteins (supplemental Fig. S1). Surface integrin levels were reduced in the ARNO knockdown MCF-7 cells (Fig. 2, A and B).
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