RESUMO
Primary cilia are sensory membrane protrusions whose dysfunction causes ciliopathies. INPP5E is a ciliary phosphoinositide phosphatase mutated in ciliopathies like Joubert syndrome. INPP5E regulates numerous ciliary functions, but how it accumulates in cilia remains poorly understood. Herein, we show INPP5E ciliary targeting requires its folded catalytic domain and is controlled by four conserved ciliary localization signals (CLSs): LLxPIR motif (CLS1), W383 (CLS2), FDRxLYL motif (CLS3) and CaaX box (CLS4). We answer two long-standing questions in the field. First, partial CLS1-CLS4 redundancy explains why CLS4 is dispensable for ciliary targeting. Second, the essential need for CLS2 clarifies why CLS3-CLS4 are together insufficient for ciliary accumulation. Furthermore, we reveal that some Joubert syndrome mutations perturb INPP5E ciliary targeting, and clarify how each CLS works: (i) CLS4 recruits PDE6D, RPGR and ARL13B, (ii) CLS2-CLS3 regulate association to TULP3, ARL13B, and CEP164, and (iii) CLS1 and CLS4 cooperate in ATG16L1 binding. Altogether, we shed light on the mechanisms of INPP5E ciliary targeting, revealing a complexity without known parallels among ciliary cargoes.
Assuntos
Ciliopatias , Doenças Renais Císticas , Anormalidades Múltiplas , Cerebelo/anormalidades , Cílios/metabolismo , Anormalidades do Olho , Proteínas do Olho/metabolismo , Humanos , Doenças Renais Císticas/genética , Doenças Renais Císticas/metabolismo , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Retina/anormalidadesRESUMO
Primary cilia are hair-like projections of the cell membrane supported by an inner microtubule scaffold, the axoneme, which polymerizes out of a membrane-docked centriole at the ciliary base. By working as specialized signaling compartments, primary cilia provide an optimal environment for many G protein-coupled receptors (GPCRs) and their effectors to efficiently transmit their signals to the rest of the cell. For this to occur, however, all necessary receptors and signal transducers must first accumulate at the ciliary membrane. Serotonin receptor 6 (HTR6) and Somatostatin receptor 3 (SSTR3) are two GPCRs whose signaling in brain neuronal cilia affects cognition and is implicated in psychiatric, neurodegenerative, and oncologic diseases. Over a decade ago, the third intracellular loops (IC3s) of HTR6 and SSTR3 were shown to contain ciliary localization sequences (CLSs) that, when grafted onto non-ciliary GPCRs, could drive their ciliary accumulation. Nevertheless, these CLSs were dispensable for ciliary targeting of HTR6 and SSTR3, suggesting the presence of additional CLSs, which we have recently identified in their C-terminal tails. Herein, we review the discovery and mapping of these CLSs, as well as the state of the art regarding how these CLSs may orchestrate ciliary accumulation of these GPCRs by controlling when and where they interact with the ciliary entry and exit machinery via adaptors such as TULP3, RABL2 and the BBSome.
Assuntos
Cílios/metabolismo , Receptores de Serotonina/metabolismo , Receptores de Somatostatina/metabolismo , Animais , Humanos , Domínios e Motivos de Interação entre Proteínas/fisiologia , Sinais Direcionadores de Proteínas/genética , Transporte Proteico/fisiologia , Receptores de Serotonina/química , Receptores de Serotonina/genética , Receptores de Somatostatina/química , Receptores de Somatostatina/genéticaRESUMO
G protein-coupled receptors (GPCRs) are the most common pharmacological target in human clinical practice. To perform their functions, many GPCRs must accumulate inside primary cilia, microtubule-based plasma membrane protrusions working as cellular antennae. Nevertheless, the molecular mechanisms underlying GPCR ciliary targeting remain poorly understood. Serotonin receptor 6 (HTR6) and somatostatin receptor 3 (SSTR3) are two brain-enriched ciliary GPCRs involved in cognition and pathologies such as Alzheimer's disease and cancer. Although the third intracellular loops (IC3) of HTR6 and SSTR3 suffice to target non-ciliary GPCRs to cilia, these IC3s are dispensable for ciliary targeting of HTR6 and SSTR3 themselves, suggesting these GPCRs contain additional ciliary targeting sequences (CTSs). Herein, we discover and characterize novel CTSs in HTR6 and SSTR3 C-terminal tails (CT). These CT-CTSs (CTS2) act redundantly with IC3-CTSs (CTS1), each being sufficient for ciliary targeting. In HTR6, RKQ and LPG motifs are critical for CTS1 and CTS2 function, respectively, whereas in SSTR3 these roles are mostly fulfilled by AP[AS]CQ motifs in IC3 and juxtamembrane residues in CT. Furthermore, we shed light on how these CTSs promote ciliary targeting by modulating binding to ciliary trafficking adapters TULP3 and RABL2.
Assuntos
Membrana Celular/metabolismo , Cílios/metabolismo , Receptores de Serotonina/química , Receptores de Serotonina/metabolismo , Receptores de Somatostatina/química , Receptores de Somatostatina/metabolismo , Transdução de Sinais/genética , Sequência de Aminoácidos , Animais , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico/genética , TransfecçãoRESUMO
Prothymosin α (ProTα) is a multifunctional protein that, in mammalian cells, is involved in nuclear metabolism through its interaction with histones and that also has a cytosolic role as an apoptotic inhibitor. ProTα is phosphorylated by a protein kinase (ProTαK), the activity of which is dependent on phosphorylation. ProTα phosphorylation also correlates with cell proliferation. Mass spectrometric analysis of ProTαK purified from human tumor lymphocytes (NC37 cells) enabled us to identify this enzyme as the M2-type isoenzyme of pyruvate kinase. A study on the relationship between ProTαK activity and pyruvate kinase isoforms in NC37 cells and in other cell types confirmed that the M2 isoform is the enzyme responsible for ProTαK activity in proliferating cells. Yet, about 10% of the cellular pool of the M2 isoform shows specific affinity for ProTα and is responsible for ProTαK activity. This pool of M2 protein possesses no observable pyruvate kinase activity and changes its responses to various effectors of pyruvate kinase activity; however, these responses to PK effectors are maintained by the main cellular fraction containing the M2 isoform. Acquisition of ProTαK activity by M2 seems to be due to the phosphorylation of serine and threonine residues, which, besides being essential for its catalytic activity, induces a trimeric association of ProTαK. This association can be shifted to a tetrameric form by fructose 1, 6-bisphosphate, which results in a decrease in ProTαK activity.