Filling GAPs in G protein- coupled receptor (GPCR)-mediated Ras adaptation and chemotaxis

ABSTRACT

Eukaryotic cells sense and migrate toward chemoattractant gradients using G protein-coupled receptor (GPCR) signaling pathways. The fascinating feature of chemotaxis is that cells migrate through chemoattractant gradients with huge concentration ranges by “adaptation.” Adaptive cells no longer respond to the present stimulus but remain sensitive to stronger stimuli, providing the fundamental strategy for chemotaxis through gradients with a broad range of concentrations. Ras activation is the first step in the GPCR-mediated chemosensing signaling pathways that displays adaptation. However, the molecular mechanism of Ras adaptation is not fully understood. Here, we highlight C2GAP1, a GPCR-activated Ras negative regulator, that locally inhibits Ras signaling for adaptation and long-range chemotaxis in D. discoideum.

Introduction

Chemotaxis is directional cell migration guided by chemoattractant gradients [1–3]. This cellular behavior plays critical roles in many physiological processes, such as neuron patterning, inflammatory responses, angiogenesis, metastasis of cancer cells, and the early development of the model organism Dictyostelium discoideum [2,4,5]. Chemotactic cells detect and respond to chemoattractants using G protein-coupled receptor (GPCR) signaling pathways. Both human neutrophils and D. discoideum cells chemotax toward their chemoattractants over an enormously broad concentration range (10⁻⁹ to 10⁻⁵ M) by applying a cellular mechanism called adaptation [6–8]. D. discoideum provides a powerful system to identify new components, develop and verify a new theory to understand the molecular mechanism underlining chemotaxis. The critical nature of adaptation is that adaptive cells no longer respond to a continuing, existing stimulus but remain responsive to stimuli at higher concentrations. This provides the fundamental ability needed for gradient sensing and directional cell migration toward the source of chemoattractant gradients.

Camp-induced Ras activation is the first step in the cAR1 GPCR signaling pathway that displays adaptation

In D. discoideum, chemoattractant cAMP binds to its receptor cAR1 and induces persistent activation of the heterotrimeric Gα2/Gβγ complex [9–13], indicating that adaptation occurs downstream of G protein activation. The free Gα2 and Gβγ subunits activate multiple downstream effectors and signaling pathways, including Ras [2,14,15]. Ras is a molecular switch cycling between active and inactive forms: it is activated by guanine nucleotide exchange factors (GEFs), which stimulate the release of guanosine diphosphate (GDP) to allow binding of guanosine triphosphate (GTP); and inactivated by GTPase-activating proteins (GAPs), which convert active RasGTP to RasGDP by stimulating their intrinsic GTPase activity. The GPCR-mediated activation profile of Ras GEFs remains largely unclear [16–18]. A robust, transient Ras activation in response to uniform cAMP stimulation is the first step in the chemoattractant GPCR-mediated signaling pathways that displays adaptation behavior [19], indicating that Ras negative regulators, such as Ras GAPs, may play a role in adaptation and chemotaxis.

Ras is activated by chemoattractant GPCR/G protein activation or associated with cytoskeletal activity

In vegetative D. discoideum cells, active Ras is often associated with pseudopod protrusion and directly mediates the processes of macropinocytosis, fluid uptake, and phagocytosis [19–22]. In starving cAMP chemotactic cells, uniformly applied cAMP stimulation triggers a transient Ras activation, followed by a small reactivation that is also associated with pseudopod protrusions and chemotaxis (Fig. 1A) [23–25]. Consistent with the above, in the cells treated with the actin polymerization inhibitor latrunculin and therefore lacking cytoskeletal activity, uniform cAMP stimulation triggered only the initial transient Ras activation, a typical adaptation behavior [19,24,26,27]. These results indicate that the second reactivation of Ras in the cells in response to uniformly applied cAMP stimulation depends on cytoskeletal activity. More importantly, we found that the adaptation profile of Ras is cAMP-concentration dependent [24]. In response to cAMP stimuli at different concentrations, Ras activation adapted to different levels after the initial, transient activation. In response to a low cAMP stimulation (10 nM), there was an almost complete return to the pre-stimulus level, designated a perfect adaptation. In response to a high cAMP stimulation (10 μM), there was an incomplete return to a higher level than the pre-stimulus baseline, designated an imperfect adaptation [24], indicating that there are more Ras proteins remaining active after adaptation in response to a stronger stimulation. This imperfect adaptation profile of the GPCR-mediated Ras adaptation is extremely important due to the fundamental role of Ras in activating downstream effectors to establish the intracellular polarized response for gradient sensing and chemotaxis.

Figure 1.

Multiple Ras GAP proteins are involved in GPCR-mediated Ras adaptation. A. GPCR-mediated Ras activation in cells with or without cytoskeletal activity. B. Both DdNF1 and C2GAP1 might be involved in GPCR-mediated Ras adaptation.

Multiple Ras GAP proteins are involved in the GPCR-mediated Ras adaptation in D. discoideum

Twelve genes encode potential Ras GAP proteins in D. discoideum. Disruption of RasGAP genes nf1 (axeB⁻ or nf1⁻), ddnf1 (ddnf1⁻ or nfaA⁻), or c2gapA (c2gapA⁻) results in enhanced Ras activity [20,24,26]. axeB⁻ cells showed increased micropinocytosis and axenic growth, indicating a leading role of axeB in the fluid uptake and axenic growth of the cells [20]. nfa⁻ cells displayed cAMP-induced prolonged, nonadaptive Ras activation and defects in chemotaxis [26]. We recently identified the c2gapA gene encoding a C2 domain-containing Ras GAP protein, C2GAP1, that is highly expressed only in the cAMP-chemotactic stage of D. discoideum cells [24]. More importantly, c2gapA⁻ cells displayed an altered profile of cAMP-induced Ras activation that is different from that of either WT or nfa⁻ cells. Compared to the rapid, transient, persistently adaptive Ras activation in WT cells (Fig. 1B), uniform cAMP stimulation triggers a stronger, longer initial Ras activation in nfa⁻ cells that gradually decreases after 20 seconds [26]. In contrast to nfa⁻ cells, in c2gapA⁻ cells, uniform cAMP stimulation also triggered the initial transient Ras adaptation. However, c2gapA⁻ cells fail to adapt persistently to cAMP stimulation [24]. Failure in persistent adaptation is also observed in c2gapA⁻ cells without cytoskeletal activity, indicating that the non-adaptive behavior of c2gapA⁻ cells is F-actin-independent and different from F-actin-dependent reactivation in WT cells. Based on the different Ras activation profiles in WT, nfa⁻, and c2gapA⁻ cells, we propose that multiple Ras GAP proteins are required for Ras adaptation and act at different steps: DdNF1 might be responsible for the quick turn-off of Ras activation at the initial step, while C2GAP1 likely plays a crucial role in persistent, long-term adaptation at the second step in the adaptation process (Fig. 1B). The key features of the membrane-targeting C2GAP1 are that it requires Ras and is chemoattractant concentration-dependent [24]. Consistently, the failure of Ras adaptation and the subsequent PIP₃ response and defects in chemotaxis in c2gapA⁻ cells are also chemoattractant concentration-dependent. Our findings uncover a molecular mechanism of Ras adaptation by which cells achieve adaptation and effective chemotaxis in response to gradients with a broad range of concentrations.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.