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Our laboratory has identified a number of small molecules that bind to G protein βγsubunits (Gβγ) by competing for peptide binding to the G βγ "hot spot." M119/Gallein were identified as inhibitors of G βγ subunit signaling. Here we examine the activity of another molecule identified in this screen, 12155, which we show that in contrast to M119/Gallein had no effect on G βγ-mediated phospholipase C or phosphoinositide 3-kinase (PI3K) γactivation in vitro. Also in direct contrast to M119/Gallein, 12155 caused receptor-independent Ca2+ release, and activated other downstream targets of G βγ including extracellular signal regulated kinase (ERK), protein kinase B (Akt) in HL60 cells differentiated to neutrophils. We show that 12155 releases G βγ in vitro from Gαi1 β1γ2 heterotrimers by causing its dissociation from G αGDP without inducing nucleotide exchange in the G α subunit. We used this novel probe to examine the hypothesis that G βγ release is sufficient to direct chemotaxis of neutrophils in the absence of receptor or G protein α subunit activation. 12155 directed chemotaxis of HL60 cells and primary neutrophils in a transwell migration assay with responses similar to those seen for the natural chemotactic peptide n-formyl-Met-Leu-Phe. These data indicate that release of free G βγ is sufficient to drive directional chemotaxis in a G protein-coupled receptor signaling-independent manner. [ABSTRACT FROM AUTHOR]