IRAG1 Deficient Mice Develop PKG1ß Dependent Pulmonary Hypertension.

PKGs are serine/threonine kinases. PKG1 has two isoforms-PKG1α and ß. Inositol trisphosphate receptor (IP3R)-associated cGMP-kinase substrate 1 (IRAG1) is a substrate for PKG1ß. IRAG1 is also known to further interact with IP3RI, which mediates intracellular Ca2+ release. However, the role of IRAG1 in PH is not known. Herein, WT and IRAG1 KO mice were kept under normoxic or hypoxic (10% O2) conditions for five weeks. Animals were evaluated for echocardiographic variables and went through right heart catheterization. Animals were further sacrificed to prepare lungs and right ventricular (RV) for immunostaining, western blotting, and pulmonary artery smooth muscle cell (PASMC) isolation. IRAG1 is expressed in PASMCs and downregulated under hypoxic conditions. Genetic deletion of IRAG1 leads to RV hypertrophy, increase in RV systolic pressure, and RV dysfunction in mice. Absence of IRAG1 in lung and RV have direct impacts on PKG1ß expression. Attenuated PKG1ß expression in IRAG1 KO mice further dysregulates other downstream candidates of PKG1ß in RV. IRAG1 KO mice develop PH spontaneously. Our results indicate that PKG1ß signaling via IRAG1 is essential for the homeostasis of PASMCs and RV. Disturbing this signaling complex by deleting IRAG1 can lead to RV dysfunction and development of PH in mice.

Pianp deficiency links GABAB receptor signaling and hippocampal and cerebellar neuronal cell composition to autism-like behavior.

Pianp (also known as Leda-1) is a type I transmembrane protein with preferential expression in the mammalian CNS. Its processing is characterized by proteolytic cleavage by a range of proteases including Adam10, Adam17, MMPs, and the γ-secretase complex. Pianp can interact with Pilrα and the GB1a subunit of the GABAB receptor (GBR) complex. A recent case description of a boy with global developmental delay and homozygous nonsense variant in PIANP supports the hypothesis that PIANP is involved in the control of behavioral traits in mammals. To investigate the physiological functions of Pianp, constitutive, global knockout mice were generated and comprehensively analyzed. Broad assessment did not indicate malformation or malfunction of internal organs. In the brain, however, decreased sizes and altered cellular compositions of the dentate gyrus as well as the cerebellum, including a lower number of cerebellar Purkinje cells, were identified. Functionally, loss of Pianp led to impaired presynaptic GBR-mediated inhibition of glutamate release and altered gene expression in the cortex, hippocampus, amygdala, and hypothalamus including downregulation of Erdr1, a gene linked to autism-like behavior. Behavioral phenotyping revealed that Pianp deficiency leads to context-dependent enhanced anxiety and spatial learning deficits, an altered stress response, severely impaired social interaction, and enhanced repetitive behavior, which all represent characteristic features of an autism spectrum disorder-like phenotype. Altogether, Pianp represents a novel candidate gene involved in autism-like behavior, cerebellar and hippocampal pathology, and GBR signaling.

Posttranslational proteolytic processing of Leda-1/Pianp involves cleavage by MMPs, ADAM10/17 and gamma-secretase.

Leda-1/Pianp is a type I transmembrane protein expressed by CNS cells, murine melanoma cell line B16F10 and rat liver sinusoidal endothelial cells. The early steps of posttranslational modifications of Leda-1/Pianp have been described to include glycosylation and processing by proprotein convertases. Here, we comprehensively characterized the subsequent steps of proteolytic processing of Leda-1/Pianp. For this purpose specific protease inhibitors and cell lines deficient in PS1, PS2, PS1/PS2 and ADAM10/17 were deployed. Leda-1/Pianp was cleaved at numerous cleavage sites within the N-terminal extracellular domain. The sheddases involved included MMPs and ADAM10/17. Ectodomain shedding yielded C-terminal fragments (CTF) of ∼15 kDa. The CTF was further processed by the γ (gamma)-secretase complex to generate the intracellular domain (ICD) of ∼10 kDa. Although PS1 was the dominant intramembrane protease, PS2 was also able to cleave Leda-1/Pianp in the absence of PS1. Thus, Leda-1/Pianp is constitutively processed by proprotein convertases, sheddases including MMPs and ADAM10/17 and intramembrane protease γ-secretase.

Leda-1/Pianp is targeted to the basolateral plasma membrane by a distinct intracellular juxtamembrane region and modulates barrier properties and E-Cadherin processing.

Leda-1/Pianp is a type-I transmembrane protein which is sorted to the basolateral membrane domain of polarized epithelial cells. Here, we investigated trafficking mechanisms and functions of Leda-1/Pianp in MDCK and MCF-7 cells. Basolateral sorting and posttranslational modifications depended on the intracellular juxtamembrane region. Functionally, Leda-1/Pianp increased the transepithelial electrical resistance generated by a polarized cell sheet. Furthermore, resistance to junctional destabilization by tumor cells was enhanced by Leda-1/Pianp indicating increased stability and tightness of intercellular junctions. While Claudin 1 and 4 expression and activities of small GTPases were not affected, γ-Secretase-mediated cleavage of E-Cadherin was attenuated by Leda-1/Pianp. Regulation of proteolytic processing is thus a molecular mechanism by which Leda-1/Pianp can affect junctional integrity and function.

Counter-regulation of the ligand-receptor pair Leda-1/Pianp and Pilrα during the LPS-mediated immune response of murine macrophages.

Liver endothelial differentiation-associated protein-1 (Leda-1/Pianp) is a type-I-transmembrane protein that is able to bind and activate immune inhibitory receptor Pilrα. Here we show that Leda-1/Pianp is strain-specifically expressed in lymphoid organs and macrophages of Th2-prone BALB/c mice but not of Th1-prone C57BL/6J mice. LPS stimulation of BALB/c bone marrow-derived macrophages (BMM) and macrophage-like Raw 264.7 cells conversely regulated Leda-1/Pianp and Pilrα expression. Pilrα induction was caused by LPS-mediated transcriptional modulation and increased mRNA expression. On the other hand, the LPS-mediated decline of Leda-1/Pianp expression was the result of proteolytic degradation by matrix metalloproteinases. In summary, these findings demonstrate that counter-regulation of the ligand-receptor pair Leda-1/Pianp and Pilrα is part of the complex innate immune response of macrophages and its genetically determined strain-specific modulation.

Proteolytic cleavage of LEDA-1/PIANP by furin-like proprotein convertases precedes its plasma membrane localization.

Liver endothelial differentiation-associated protein-1 (LEDA-1/PIANP) is a type-I-transmembrane protein first identified by us as a putative junctional protein in liver sinusoidal endothelial cells. Others have shown that LEDA-1/PIANP binds and activates immune inhibitory receptor PILRα in trans, a process that requires sialidation of LEDA-1/PIANP. Here we show that LEDA-1/PIANP is subject to O-glycosylation and sialidation as demonstrated in brain tissue as well as in LEDA-1 expressing cell lines by using anti-LEDA-1/PIANP C-terminal antibodies. In addition, analysis of LEDA-1/PIANP processing with His-tags inserted at different positions in the extracellular domain revealed that multiple steps of proteolytic cleavage occur during maturation of the protein. Proteolytic cleavage between aa59 and aa83 preceded sorting of the protein to the plasma membrane. Deletion of aa75-79 and inhibition with Furin inhibitor I confirmed that LEDA-1/PIANP is processed by a Furin-like proprotein convertase. In summary, these findings show that Furin-like proprotein convertase-dependent processing precedes plasma membrane localization of LEDA-1/PIANP that is a pre-requisite of functional receptor-ligand interactions in vitro and in vivo.