Recycling of internalized integrins is a crucial step in adhesion remodeling and 
cell movement. Recently, we determined that the ADP-ribosylation factor-guanine 
nucleotide exchange factors (ARF-GEFs) cytohesin 2/ARNO and cytohesin 3/GRP1 
have opposing effects on adhesion and stimulated β1 integrin recycling even 
though they are very closely related proteins (80% sequence identity). We have 
now determined the sequence differences underlying the differential actions of 
cytohesin 2/ARNO and cytohesin 3/GRP1. We found that the ability of cytohesins 
to promote β1 integrin recycling and adhesion depends upon the presence or 
absence of a key glycine residue in their pleckstrin homology (PH) domains. This 
glycine residue determines the phosphoinositide specificity and affinity of 
cytohesin PH domains. Switching the number of glycines in the PH domains of 
cytohesin 2 and cytohesin 3 is sufficient to reverse their effects on adhesion 
and spreading and to reverse their subcellular locations. Importantly, we also 
find that a mutant form of cytohesin 3/GRP1 that has three rather than two 
glycines in its PH domain rescues β1 integrin recycling in cytohesin 2/ARNO 
knockdown cells. Conversely, a mutant form of cytohesin 2/ARNO with two glycines 
in its PH domain fails to rescue β1 integrin recycling. Therefore, we conclude 
that phosphoinositide specificity is the sole functional difference that 
determines which cytohesin can promote integrin recycling.