PI3Kγ adapter subunits in mast cells

Abstract

The incidence of allergy and related diseases such as asthma increased rapidly over the last century, especially in western societies. The allergic symptoms impair the quality of life of patients and can even be life threatening. The symptoms vary in strength and location, leading to sneezing, allergic sinusitis, itching, broncho-constriction, diarrhoea or anaphylaxis. Allergy is an acquired hyperreactivity of the immune system towards non-harmful substances, which leads to the activation of mast cells as a central component in the disease development and progression. Thus a detailed understanding of how mast cells are activated could be beneficial for the development of novel anti-allergic medication. For proper mast cell differentiation, proliferation, tissue distribution and their activation the lipid kinase family of Phosphoinositide 3-kinase (PI3K) has been proven fundamental, which are thus putative pharmacologic targets. Class I PI3K activation by cell surface receptors facilitates the production of the lipid second messenger phosphatidylinositol(3,4,5)-trisphosphate (PtdIns(3,4,5)P3) which is central for the integration of effector proteins, equipped with PtdIns(3,4,5)P3 binding PH domains. In turn activation of serine/threonine kinases, tyrosine kinases, GEFs and GAPs of small GTPases modulate mast cell survival, proliferation, migration and degranulation. Subclass IA PI3K serves receptor tyrosine kinases (RTK) and non-receptor tyrosine kinase (NRTK) as well as immunoglobulin receptors by direct or indirect association, through their p85 family of regulatory subunits. In contrast the only class IB member, PI3Kγ, is primarily activated downstream of G-protein coupled receptors (GPCR). Thereby PI3Kγ interaction with released Gβγ subunits from heterotrimeric G proteins is required to transmit signals from the extracellular milieu. PI3Kγ is a heterodimeric complex, consisting of the catalytic subunit (p110γ) and a adapter subunit, either p101 or the recently identified p84 (or p87 PIKAP ). In line with PI3Kγ’s predominant expression in immune cells, genetic ablation of the catalytic subunit p110γ causes defects in inflammatory and allergic responses, such as leukocyte chemotaxis, mast cell migration, and the synergistic amplification of IgE and antigen mediated degranulation. While the requirement of class IB PI3K activity in mast cell function was proven in murine disease models, there is still a lack of knowledge on the physiologic function of its adapter subunits, dubbed p101 and p84. Based on in vitro experiments both adapters support p110γ activity by sensitizing p110γ for the Gβγmediated activation downstream of GPCRs. Expression analysis performed here determined mast cells most appropriate to study the physiologic requirement of p84. Mast cells have high abundance of p84 and p110γ protein, while p101 protein was undetectable. In particular the observed destabilization of p84 protein in p110γ null mast cells was beneficial to conduct elegant complementation experiments. To this end, p84 complexed with the catalytic subunit p110γ (p84:p110γ), was essential for all PI3Kγ dependent cell responses, such as adenosine driven PtdIns(3,4,5)P3 production, phosphorylation of PKB/Akt, cell migration and the adenosine enforced degranulation. Of note, increased abundance of p110γ was ineffective in compensating lack of p84. Moreover, the replacement of p84 by p101 in complex with p110γ (p101:p110γ) unraveled a non-redundant function for the two adapter subunits, as p101:p110γ failed to support degranulation, while cell migration and phosphorylation of PKB/Akt were intact. A possible explanation was provided by adapter dependent spatiotemporal differences of PtdIns(3,4,5)P3 production. Both PI3Kγ complexes produced PtdIns(3,4,5)P3 at the plasma membrane, which was however rapidly endocytosed via microtubule dependent process, when derived from p101:p110γ signaling. Especially during co-stimulation with adenosine and IgE and antigen, p84:p110γ derived PtdIns(3,4,5)P3 significantly prolonged localization to the plasma membrane was observed as compared to PtdIns(3,4,5)P3 of p101:p110γ origin. Moreover the two PI3Kγ complexes have differential sensitivity to cholesterol depleting agents. Altogether this implies adapter dependent production of distinct pools of PtdIns(3,4,5)P3 at the plasma membrane, eliciting specific cell responses. Thus it is exclusively the p84:p110γ complex, that amplifies mast degranulation and allergic responses. This adds an additional pathway for specific treatment of allergic diseases, e.g. by particular disruption of this complex.