Heterotrimeric G-protein shuttling via Gip1 extends the dynamic range of eukaryotic chemotaxis Yoichiro Kamimuraa,b,1, Yukihiro Miyanagaa,b,1, and Masahiro Uedaa,b,2 aLaboratory for Cell Signaling Dynamics, Quantitative Biology Center (QBiC), RIKEN, Suita, Osaka, 565-0874, Japan; and bLaboratory for Single Molecular Biology, Department of Biological Sciences, Graduate School of Science, Osaka University, Toyonaka, Osaka, 560-0043, Japan Edited by Peter N. Devreotes, The Johns Hopkins University School of Medicine, Baltimore, MD, and approved March 7, 2016 (received for review September 11, 2015) Chemotactic eukaryote cells can sense chemical gradients over a adaptively over a wide range in the signal transduction cascades wide range of concentrations via heterotrimeric G-protein signaling; upstream of STEN. however, the underlying wide-range sensing mechanisms are only Insight into this question is provided by bacterial chemotaxis partially understood. Here we report that a novel regulator of G pro- and other sensory systems, such as photoreceptor rhodopsin (8). teins, G protein-interacting protein 1 (Gip1), is essential for extending Chemoreceptor methylation in bacteria confers a broad chemo- the chemotactic range of Dictyostelium cells. Genetic disruption of tactic range (11). In light adaptation, the phosphorylation of rho- Gip1 caused severe defects in gradient sensing and directed cell mi- dopsins in the visual system leads to rhodopsin down-regulation by gration at high but not low concentrations of chemoattractant. Also, arrestin, which blocks physical interaction with G-protein trans- Gip1 was found to bind and sequester G proteins in cytosolic pools. ducin (12). Phosphorylation-dependent receptor internalization is a Receptor activation induced G-protein translocation to the plasma feature of other systems for suppressing intracellular responses membrane from the cytosol in a Gip1-dependent manner, causing a (13). Overall, in these sensory systems, the chemical modifications biased redistribution of G protein on the membrane along a chemo- of receptors are important for regulating the dynamic range of the attractant gradient. These findings suggest that Gip1 regulates response. Consistently, Dictyostelium cells expressing unphosphory- G-protein shuttling between the cytosol and the membrane to ensure lated mutant cAR1 exhibit a narrow chemotactic range (14), and the availability and biased redistribution of G protein on the mem- phosphorylated cAR1s have reduced affinity for cAMP (15). Thus, brane for receptor-mediated chemotactic signaling. This mechanism chemical modifications of chemoattractant receptors are also im- offers an explanation for the wide-range sensing seen in eukaryotic portant in eukaryotic chemotaxis as a mechanism to extend the chemotaxis. chemotactic range. In addition to the receptor modifications, G proteins are phosphorylated and recruited from the cytosol to the eukaryotic chemotaxis | gradient sensing | dynamic range extension | plasma membrane upon receptor stimulation in Dictyostelium cells heterotrimeric G protein (16, 17), although the relevance of these actions on wide-range sensing and adaptation is unknown. hemotaxis in eukaryotic cells is observed in many physio- Here we report that a novel regulator of G proteins, G protein- Clogical processes including embryogenesis, neuronal wiring, interacting protein 1 (Gip1), is essential for the wide-range che- wound healing, and immune responses (1, 2). Chemotactic cells motaxis in Dictyostelium cells. Gip1 regulates G-protein localiza- share basic properties including high sensitivity to shallow gradients tion between the cytosol and plasma membrane upon receptor and responsiveness to a wide dynamic range of chemoattractants activation, which targets cytosolic G proteins to the membrane in (3, 4). For instance, human neutrophils and Dictyostelium cells can a biased manner along chemoattractant gradients at higher sense spatial differences in chemoattractant concentration across chemotactic ranges. These findings provide evidence for a wide- the cell body in shallow gradients as low as 2% and exhibit che- range sensing mechanism in which Gip1-dependent G-protein motaxisovera105–106-fold range of background concentrations – (5 7). Thus, wide-range sensing and adaptation are critical features Significance of chemotaxis as well as other sensory systems such as visual signal transduction (8). However, the underlying regulatory mechanisms Eukaryotic chemotactic cells can recognize chemical gradients in eukaryotic chemotaxis remain unclear. over a wide range of concentrations. This ability is physiolog- The molecular mechanisms of chemotaxis are evolutionarily ically important for numerous biological processes; however, conserved among many eukaryotes that use G protein-coupled its underlying mechanism is unknown. Here we report that the receptors (GPCRs) and heterotrimeric G proteins to detect dynamic range of chemotaxis is extended to higher concen- chemoattractant gradients (3, 4). In Dictyostelium cells, extracellular trations by gradient sensing achieved via regulation of trimeric cAMP works as a chemoattractant, and binding to its receptor cyclic α βγ G-protein shuttling between the cytosol and plasma mem- AMP receptor 1 (cAR1) activates G proteins (G 2G ) along the brane. G protein-interacting protein 1 (Gip1) regulates this in- concentration gradient, leading to the activation of multiple sig- tracellular G-protein translocation, which redistributes cytosolic – – – naling cascades including the PI3K PTEN, TorC2 PDK PKB, G proteins to the plasma membrane along chemical gradients at phospholipase A2, and guanylyl cyclase pathways. In contrast to the high chemoattractant concentrations. This dynamic spatiotemporal spatial distributions of cAMP/cAR1 association and G-protein ac- regulation of trimeric G protein yields proper processing of tivation, downstream signaling pathways are activated in an ex- receptor-mediated signaling. tremely biased manner at the anterior or posterior of the cell (3, 4). For example, localized patches of phosphatidylinositol 3,4,5- Author contributions: Y.K., Y.M., and M.U. designed research; Y.K. and Y.M. performed research; Y.K. and Y.M. analyzed data; and Y.K., Y.M., and M.U. wrote the paper. trisphosphate (PIP3) are generated at the plasma membrane by an intracellular signal transduction excitable network (STEN) and The authors declare no conflict of interest. function as a cue to control the pseudopod formation of motile cells This article is a PNAS Direct Submission. (9,10).BecausePIP3 patches have a relatively constant size of a few 1Y.K. and Y.M. contributed equally to this work. microns in diameter, this excitable mechanism can ensure a con- 2To whom correspondence should be addressed. Email: [email protected]. stant output of chemotactic responses over a wide range of concen- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. trations. However, it is unclear how chemical gradients are sensed 1073/pnas.1516767113/-/DCSupplemental. 4356–4361 | PNAS | April 19, 2016 | vol. 113 | no. 16 www.pnas.org/cgi/doi/10.1073/pnas.1516767113 Downloaded by guest on September 23, 2021 translocation ensures the availability and biased redistribution of The interaction between Gip1 and Gβ was verified by a pull- G protein for receptor-mediated signaling at the higher range, down assay using GFP-Flag–tagged Gip1 (Gip1-GFPF), as shown which is in contrast to the chemical modification mechanisms in Fig. 1B. To identify the interaction region, the N-terminal PH underlying adaptation by sensory receptors. domain (amino acids 1–109) and the Gip1 C terminus (amino acids 108–310) were separately expressed and used in the assay. The C Results but not N terminus bound to Gβ as efficiently as the full-length Gip1 Is an Interactor of Trimeric G Protein. We identified Gip1 by protein (Fig. 1C). Furthermore, to determine whether G proteins using a tandem affinity purification (TAP) tag of Gβ.FortheTAP bind Gip1, the proteins that copurified with Gip1-GFPF by TAP assay, whole-cell extracts (WCEs) were prepared from cells in were analyzed by MS (Fig. S1 D and E). The two prominent bands β α vegetative growth and chemotactically competent cells with or at 35 and 40 kDa in Fig. S1D were G and G subtypes, re- spectively, including Gα4, Gα9, Gα5, and Gα2. Together with the without cAMP stimulation. Under these conditions, four bands – were observed at around 240, 40, 30, and 9 kDa in addition to the additional data in SI Text (Fig. S1 F K), these results confirmed that Gip1 binds to G proteins via the C terminus. TAP-tagged Gβ protein (Fig. 1A and Fig. S1A). Mass spectrometry The physiological roles of Gip1 in the development of Dictyos- (MS) analysis revealed that p40 and p9 were Gα4andGγ,re- β telium cells were investigated by examining the phenotype of gip1- spectively; p240 was ElmoE, which is a known G -binding protein knockout (gip1Δ) cells (Fig. S1L). Wild-type (WT) cells formed (18); and p30 was a previously uncharacterized protein encoded by streams that consisted of a collective migration of chemotactic cells DDB_G0271086 and designated as Gip1. Gip1 contained a pleckstrin upon starvation, whereas gip1Δ cells caused smaller aggregates with homology (PH) domain at the N terminus between amino acids reduced stream formation (Fig. 1D). Moreover, Gip1 over- 1–109 and an unidentified region at the C terminus. Gip1 homo- expression in WT cells (Gip1OE cells) delayed progression of