G protein selectivity profile of GPR56/ADGRG1 and its effect on downstream effectors.

GPR56, an adhesion G-protein coupled receptor (aGPCRs) with constitutive and ligand-promoted activity, is involved in many physiological and pathological processes. Whether the receptor's constitutive or ligand-promoted activation occur through the same molecular mechanism, and whether different activation modes lead to functional selectivity between G proteins is unknown. Here we show that GPR56 constitutively activates both G12 and G13. Unlike constitutive activation and activation with 3-α-acetoxydihydrodeoxygedunin (3αDOG), stimulation with an antibody, 10C7, directed against GPR56's extracellular domain (ECD) led to an activation that favors G13 over G12. An autoproteolytically deficient mutant, GPR56-T383A, was also activated by 10C7 indicating that the tethered agonist (TA) exposed through autocatalytic cleavage, is not required for this activation modality. In contrast, this proteolysis-resistant mutant could not be activated by 3αDOG indicating different modes of activation by the two ligands. We show that an N-terminal truncated GPR56 construct (GPR56-Δ1-385) is devoid of constitutive activity but was activated by 3αDOG. Similarly to 3αDOG, 10C7 promoted the recruitment of ß-arrestin-2 but GPR56 internalization was ß-arrestin independent. Despite the slow activation mode of 10C7 that favors G13 over G12, it efficiently activated the downstream Rho pathway in BT-20 breast cancer cells. These data show that different GPR56 ligands have different modes of activation yielding differential G protein selectivity but converging on the activation of the Rho pathway both in heterologous expressions system and in cancer cells endogenously expressing the receptor. 10C7 is therefore an interesting tool to study both the processes underlying GPR56 activity and its role in cancer cells.

G protein selectivity profile of GPR56/ADGRG1 and its effect on downstream effectors.

GPR56, an adhesion G-protein coupled receptor (aGPCRs) with constitutive and ligand-promoted activity, is involved in many physiological and pathological processes. Whether the receptor's constitutive or ligand-promoted activation occur through the same molecular mechanism, and whether different activation modes lead to functional selectivity between G proteins is unknown. Here we show that GPR56 constitutively activates both G12 and G13. Unlike constitutive activation and activation with 3-a-acetoxydihydrodeoxygedunin (3αDOG), stimulation with an antibody, 10C7, directed against GPR56's extracellular domain (ECD) led to an activation that favors G13 over G12. An autoproteolytically deficient mutant, GPR56-T383A, was also activated by 10C7 indicating that the tethered agonist (TA) exposed through autocatalytic cleavage, is not required for this activation modality. In contrast, this proteolysis-resistant mutant could not be activated by 3αDOG indicating different modes of activation by the two ligands. We show that an N-terminal truncated GPR56 construct (GPR56-Δ1-385) is devoid of constitutive activity but was activated by 3αDOG. Similarly to 3αDOG, 10C7 promoted the recruitment of b-arrestin-2 but GPR56 internalization was ß-arrestin independent. Despite the slow activation mode of 10C7 that favors G13 over G12, it efficiently activated the downstream Rho pathway in BT-20 breast cancer cells. These data show that different GPR56 ligands have different modes of activation yielding differential G protein selectivity but converging on the activation of the Rho pathway both in heterologous expressions system and in cancer cells endogenously expressing the receptor. 10C7 is therefore an interesting tool to study both the processes underlying GPR56 activity and its role in cancer cells.

Primary Cilium Identifies a Quiescent Cell Population in the Human Intestinal Crypt.

Primary cilia are sensory antennae located at the cell surface which mediate a variety of extracellular signals involved in development, tissue homeostasis, stem cells and cancer. Primary cilia are found in an extensive array of vertebrae cells but can only be generated when cells become quiescent. The small intestinal epithelium is a rapidly self-renewing tissue organized into a functional unit called the crypt-villus axis, containing progenitor and differentiated cells, respectively. Terminally differentiated villus cells are notoriously devoid of primary cilia. We sought to determine if intestinal crypts contain a quiescent cell population that could be identified by the presence of primary cilia. Here we show that primary cilia are detected in a subset of cells located deep in the crypts slightly above a Paneth cell population. Using a normal epithelial proliferative crypt cell model, we show that primary cilia assembly and activity correlate with a quiescent state. These results provide further evidence for the existence of a quiescent cell population in the human small intestine and suggest the potential for new modes of regulation in stem cell dynamics.

Characterization of PGua4, a Guanidinium-Rich Peptoid that Delivers IgGs to the Cytosol via Macropinocytosis.

To investigate the structure-cellular penetration relationship of guanidinium-rich transporters (GRTs), we previously designed PGua4, a five-amino acid peptoid containing a conformationally restricted pattern of eight guanidines, which showed high cell-penetrating abilities and low cell toxicity. Herein, we characterized the cellular uptake selectivity, internalization pathway, and intracellular distribution of PGua4, as well as its capacity to deliver cargo. PGua4 exhibits higher penetration efficiency in HeLa cells than in six other cell lines (A549, Caco-2, fibroblast, HEK293, Mia-PaCa2, and MCF7) and is mainly internalized by clathrin-mediated endocytosis and macropinocytosis. Confocal microscopy showed that it remained trapped in endosomes at low concentrations but induced pH-dependent endosomal membrane destabilization at concentrations ≥10 µM, allowing its diffusion into the cytoplasm. Importantly, PGua4 significantly enhanced macropinocytosis and the cellular uptake and cytosolic delivery of large IgGs following noncovalent complexation. Therefore, in addition to its peptoid nature conferring high resistance to proteolysis, PGua4 presents characteristics of a promising tool for IgG delivery and therapeutic applications.

Matriptase processing of APLP1 ectodomain alters its homodimerization.

The amyloid beta peptide (Aß) is derived from the amyloid precursor protein (APP) by secretase processing. APP is also cleaved by numerous other proteases, such as the type II transmembrane serine protease matriptase, with consequences on the production of Aß. Because the APP homolog protein amyloid-like protein 1 (APLP1) shares similarities with APP, we sought to determine if matriptase also plays a role in its processing. Here, we demonstrate that matriptase directly interacts with APLP1 and that APLP1 is cleaved in cellulo by matriptase in its E1 ectodomains at arginine 124. Replacing Arg124 with Ala abolished APLP1 processing by matriptase. Using a bioluminescence resonance energy transfer (BRET) assay we found that matriptase reduces APLP1 homodimeric interactions. This study identifies matriptase as the first protease cleaving APLP1 in its dimerization domain, potentially altering the multiple functions associated with dimer formation.

Combining RNAi and Immunofluorescence Approaches to Investigate Post-endocytic Sorting of GPCRs into Multivesicular Bodies.

Following stimulation, G protein-coupled receptors (GPCRs) are internalized and transported to early endosomes where they are either recycled back to the plasma membrane for another round of activation or targeted to the lysosomes for degradation and long-term signal termination. This latter requires internalization of receptors into intraluminal vesicles (ILVs) of multivesicular bodies (MVBs) for complete degradation following fusion with lysosomes. This endosomal sorting step is highly regulated and has profound functional consequences. This chapter describes how RNAi and confocal microscopy methods can be combined to evaluate whether a protein of interest (herein Gαs) is involved in GPCR sorting into ILVs of MVBs.