Expression and immunoaffinity purification of recombinant soluble human GPR56 protein for the analysis of GPR56 receptor shedding by ELISA

Yang, Tai-Yun; Chiang, Nien‐Yi; Tseng, Wen-Yi; Pan, Hsiao-Lin; Peng, Yen-Ming; Shen, Jiann-Jong; Wu, Kun; Kuo, Ming‐Ling; Chang, Gin-Wen; Lin, Hsi‐Hsien

doi:10.1016/j.pep.2014.11.013

Cited by 9 publications

(9 citation statements)

References 42 publications

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“…Agonistic antibodies directed to GPR56 have been reported but their mechanisms of action are not fully understood (Ohta et al, 2015, Yang et al, 2015). The lack of well-characterized agonists and antagonists has hampered mechanistic studies of GPR56 and other aGPCRs.…”

Section: Discussionmentioning

confidence: 99%

Structural Basis for Regulation of GPR56/ADGRG1 by Its Alternatively Spliced Extracellular Domains

Salzman¹,

Ackerman

Ding

et al. 2016

Neuron

100

181

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Summary Adhesion G-protein-coupled receptors (aGPCRs) play critical roles in diverse neurobiological processes including brain development, synaptogenesis, and myelination. aGPCRs have large alternatively spliced extracellular regions (ECRs) that likely mediate intercellular signaling; however, the precise roles of ECRs remain unclear. The aGPCR GPR56/ADGRG1 regulates both oligodendrocyte and cortical development. Accordingly, human GPR56 mutations cause myelination defects and brain malformations. Here, we determined the crystal structure of the GPR56 ECR, the first structure of any complete aGPCR ECR, in complex with an inverse-agonist monobody, revealing a GPCR-Autoproteolysis-Inducing domain and a previously unidentified domain that we term Pentraxin/Laminin/neurexin/sex-hormone-binding-globulin-Like (PLL). Strikingly, PLL domain deletion caused increased signaling and characterizes a GPR56 splice variant. Finally, we show that an evolutionarily conserved residue in the PLL domain is critical for oligodendrocyte development in vivo. Thus, our results suggest that the GPR56 ECR has unique and multifaceted regulatory functions, providing novel insights into aGPCR roles in neurobiology.

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Section: Discussionmentioning

confidence: 99%

Structural Basis for Regulation of GPR56/ADGRG1 by Its Alternatively Spliced Extracellular Domains

Salzman¹,

Ackerman

Ding

et al. 2016

Neuron

100

181

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“…The powerful combination of ECR-targeted synthetic proteins, including monobodies and antibodies (35,40,51), with 7TM-targeted small molecule ligands will be invaluable in future mechanistic and pharmacological studies of aGPCRs.…”

Section: Discussionmentioning

confidence: 99%

Stachel -independent modulation of GPR56/ADGRG1 signaling by synthetic ligands directed to its extracellular region

Salzman

Zhang

Gupta

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Adhesion G protein-coupled receptors (aGPCRs) play critical roles in diverse biological processes, including neurodevelopment and cancer progression. aGPCRs are characterized by large and diverse extracellular regions (ECRs) that are autoproteolytically cleaved from their membrane-embedded signaling domains. Although ECRs regulate receptor function, it is not clear whether ECRs play a direct regulatory role in G-protein signaling or simply serve as a protective cap for the activating "Stachel" sequence. Here, we present a mechanistic analysis of ECR-mediated regulation of GPR56/ADGRG1, an aGPCR with two domains [pentraxin and laminin/neurexin/sex hormonebinding globulin-like (PLL) and G protein-coupled receptor autoproteolysis-inducing (GAIN)] in its ECR. We generated a panel of high-affinity monobodies directed to each of these domains, from which we identified activators and inhibitors of GPR56-mediated signaling. Surprisingly, these synthetic ligands modulated signaling of a GPR56 mutant defective in autoproteolysis and hence, in Stachel peptide exposure. These results provide compelling support for a ligandinduced and ECR-mediated mechanism that regulates aGPCR signaling in a transient and reversible manner, which occurs in addition to the Stachel-mediated activation.adhesion GPCR | allostery | cell signaling | monobody | protein engineering

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“…Shedding is likely to generate distinct soluble receptor fragments that have different cellular functions from the membrane-bound proteins. Previously, we and others have detected the presence of soluble GPR56 (sGPR56; the NTF that has been shed from the membrane) in transfected cells, suggesting constitutive GPR56 shedding (Chiang et al, 2011;Jin et al, 2007;Yang et al, 2015). More intriguingly, expression of a truncated GPR56 receptor that lacked the NTF was shown to signal constitutively (Paavola et al, 2011).…”

Section: Introductionmentioning

confidence: 91%

“…DNA and protein reagents were obtained from Invitrogen (Carlsbad, CA), Qiagen (Valencia, CA) and Amersham (GE Healthcare). The CG-2 and CG-3 monoclonal antibodies (mAb) against GPR56 have been described previously (5 µg/ml) (Yang et al, 2015). FITC-conjugated goat anti-mouse IgG was from Jackson ImmunoResearch (West Grove, PA).…”

Section: General Reagents and Cell Culturementioning

confidence: 99%

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Heparin interacts with adhesion-GPCR GPR56/ADGRG1, reduces receptor shedding, and promotes cell adhesion and motility

Chiang

Chang

Huang

et al. 2016

Journal of Cell Science

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GPR56 is an adhesion-class G-protein-coupled receptor responsible for bilateral frontoparietal polymicrogyria (BFPP), a severe disorder of cortical formation. Additionally, GPR56 is involved in biological processes as diverse as hematopoietic stem cell generation and maintenance, myoblast fusion, muscle hypertrophy, immunoregulation and tumorigenesis. Collagen III and tissue transglutaminase 2 (TG2) have been revealed as the matricellular ligands of GPR56 involved in BFPP and melanoma development, respectively. In this study, we identify heparin as a glycosaminoglycan interacting partner of GPR56. Analyses of truncated and mutant GPR56 proteins reveal two basicresidue-rich clusters, R , as the major heparin-interacting motifs that overlap partially with the collagen III-and TG2-binding sites. Interestingly, the GPR56-heparin interaction is modulated by collagen III but not TG2, even though both ligands are also heparin-binding proteins. Finally, we show that the interaction with heparin reduces GPR56 receptor shedding, and enhances cell adhesion and motility. These results provide novel insights into the interaction of GPR56 with its multiple endogenous ligands and have functional implications in diseases such as BFPP and cancer.

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Expression and immunoaffinity purification of recombinant soluble human GPR56 protein for the analysis of GPR56 receptor shedding by ELISA

Cited by 9 publications

References 42 publications

Structural Basis for Regulation of GPR56/ADGRG1 by Its Alternatively Spliced Extracellular Domains

Structural Basis for Regulation of GPR56/ADGRG1 by Its Alternatively Spliced Extracellular Domains

Stachel -independent modulation of GPR56/ADGRG1 signaling by synthetic ligands directed to its extracellular region

Heparin interacts with adhesion-GPCR GPR56/ADGRG1, reduces receptor shedding, and promotes cell adhesion and motility

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