36 research outputs found
Die Physiologische Relevanz des G-Protein-gekoppelten Rezeptors GPR34
Die Familie der G-Protein-gekoppelten Rezeptoren (GPCRs) bildet die gröĂte Gruppe von Membranrezeptoren im menschlichen Organismus. FĂŒr viele GPCRs sind bisher die physiologischen Funktionen nicht bekannt. Das biologische VerstĂ€ndnis der Funktionen im menschlichen Organismus dieser sogenannten âorphanâ GPCRs (oGPCRs) hat, aufgrund möglicher kausaler Beteiligung an der Pathogenese von Erkrankungen sowie deren therapeutische Beeinflussbarkeit, hohe medizinische Relevanz.
Die GPCRs der P2Y12-Ă€hnliche Rezeptorgruppe besitzen eine groĂe physiologische Bedeutung bei der Thrombozytenaggregation und der Induktion der Migration von immunokompetenten Zellen in SchĂ€digungsgebiete. Der ADP-Rezeptor P2Y12 kann durch verschiedene pharmakologische Wirkstoffe beeinflusst werden, was bereits klinisch-therapeutisch genutzt wird. Diese Gruppe von GPCRs enthĂ€lt jedoch auch Mitglieder, deren Funktionen völlig unbekannt sind. Einer dieser oGPCRs ist der GPR34. Ziel dieser Arbeit war es, mittels verschiedener in-vitro-Methoden und anhand eines GPR34-defizienten Mausstamms die physiologische Relevanz dieses P2Y12-Ă€hnlichen Rezeptors zu analysieren. Dazu wurde ein GPR34-Knockout-Mausmodell etabliert. Die GPR34-Defizienz hatte keinen wesentlichen Einfluss auf die Entwicklung, Morphologie, das Wachstum oder die FertilitĂ€t bei MĂ€usen. Die Ergebnisse aus Immunisierungsâ und Infektionsstudien zeigten jedoch, dass dieser evolutionĂ€r hoch konservierte Rezeptor eine wichtige Funktion in der Feinkontrolle der zellulĂ€ren Immunabwehr ausĂŒbt. Neben einer verstĂ€rkten Antwort im Delayed-type Hypersensitivity (DTH)-Test war die Abwehr einer Cryptococcus-Infektion in diesem GPR34-defizienten Tiermodell beeintrĂ€chtigt. Signifikant erhöhte Zytokinspiegel nach Antigen- bzw. Pathogenexposition deuteten auf eine gestörte Immunregulation in GPR34-defizienten MĂ€usen hin. WeiterfĂŒhrende Untersuchungen sollten sich der Identifizierung des endogenen Agonisten und der Funktion des GPR34 bei der Koordinierung der zellulĂ€ren Immunreaktion widmen
Functional relevance of naturally occurring mutations in adhesion G protein-coupled receptor ADGRD1 (GPR133)
Background: A large number of human inherited and acquired diseases and phenotypes are caused by mutations in G protein-coupled receptors (GPCR). Genome-wide association studies (GWAS) have shown that variations in the ADGRD1 (GPR133) locus are linked with differences in metabolism, human height and heart frequency. ADGRD1 is a Gs protein-coupled receptor belonging to the class of adhesion GPCRs. Results: Analysis of more than 1000 sequenced human genomes revealed approximately 9000 single nucleotide polymorphisms (SNPs) in the human ADGRD1 as listed in public data bases. Approximately 2.4 % of these SNPs are located in exons resulting in 129 non-synonymous SNPs (nsSNPs) at 119 positions of ADGRD1. However, the functional relevance of those variants is unknown. In-depth characterization of these amino acid changes revealed several nsSNPs (A448D, Q600stop, C632fs [frame shift], A761E, N795K) causing full or partial loss of receptor function, while one nsSNP (F383S) significantly increased basal activity of ADGRD1. Conclusion: Our results show that a broad spectrum of functionally relevant ADGRD1 variants is present in the human population which may cause clinically relevant phenotypes, while being compatible with life when heterozygous
Gpr126 functions in schwann cells to control differentiation and myelination via G-protein activation
The myelin sheath surrounding axons ensures that nerve impulses travel quickly and efficiently, allowing for the proper function of the vertebrate nervous system. We previously showed that the adhesion G-protein-coupled receptor (aGPCR) Gpr126 is essential for peripheral nervous system myelination, although the molecular mechanisms by which Gpr126 functions were incompletely understood. aGPCRs are a significantly understudied protein class, and it was unknown whether Gpr126 couples to G-proteins. Here, we analyze Dhh(Cre);Gpr126(fl/fl) conditional mutants, and show that Gpr126 functions in Schwann cells (SCs) for radial sorting of axons and myelination. Furthermore, we demonstrate that elevation of cAMP levels or protein kinase A activation suppresses myelin defects in Gpr126 mouse mutants and that cAMP levels are reduced in conditional Gpr126 mutant peripheral nerve. Finally, we show that GPR126 directly increases cAMP by coupling to heterotrimeric G-proteins. Together, these data support a model in which Gpr126 functions in SCs for proper development and myelination and provide evidence that these functions are mediated via G-protein-signaling pathways
The N Terminus of Adhesion G ProteinâCoupled Receptor GPR126/ ADGRG6 as Allosteric Force Integrator
The adhesion G proteinâcoupled receptor (aGPCR) GPR126/ADGRG6 plays an important
role in several physiological functions, such as myelination or peripheral nerve repair. This
renders the receptor an attractive pharmacological target. GPR126 is a mechano-sensor
that translates the binding of extracellular matrix (ECM) molecules to its N terminus into a
metabotropic intracellular signal. To date, the structural requirements and the character of
the forces needed for this ECM-mediated receptor activation are largely unknown. In this
study, we provide this information by combining classic second-messenger detection with
single-cell atomic force microscopy. We established a monoclonal antibody targeting the N
terminus to stimulate GPR126 and compared it to the activation through its known ECM
ligands, collagen IV and laminin 211. As each ligand uses a distinct mode of action, the N
terminus can be regarded as an allosteric module that can fine-tune receptor activation in a
context-specific manner
Mechano-Dependent Phosphorylation of the PDZ-Binding Motif of CD97/ADGRE5 Modulates Cellular Detachment
Summary Cells respond to mechanical stimuli with altered signaling networks. Here, we show that mechanical forces rapidly induce phosphorylation of CD97/ADGRE5 (pCD97) at its intracellular C-terminal PDZ-binding motif (PBM). Biochemically, this phosphorylation disrupts CD97 binding to PDZ domains of the scaffold protein DLG1. In shear-stressed cells, pCD97 appears not only in junctions, retracting fibers, and the attachment area but also in lost membrane patches, demonstrating (intra)cellular detachment at the CD97 PBM. This motif is critical for the CD97-dependent mechanoresponse. Cells expressing CD97 without the PBM are more deformable, and under shear stress, these cells lose cell contacts faster and show changes in the actin cytoskeleton when compared with cells expressing full-length CD97. Our data indicate CD97 linkage to the cytoskeleton. Consistently, CD97 knockout phenocopies CD97 without the PBM, and membranous CD97 is organized in an F-actin-dependent manner. In summary, CD97 shapes the cellular mechanoresponse through signaling modulation via its PBM
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Elevated expression of the adhesion GPCR ADGRL4/ELTD1 promotes endothelial sprouting angiogenesis without activating canonical GPCR signalling.
Funder: Rhodes Scholarships; doi: http://dx.doi.org/10.13039/501100000697Funder: Clarendon ScholarshipFunder: Ernest Oppenheimer Memorial Trust; doi: http://dx.doi.org/10.13039/501100009978Funder: European Union (European Social Fund)Funder: Free State of SaxonyFunder: Cancer Research UK; doi: http://dx.doi.org/10.13039/501100000289Funder: Breast Cancer Research Foundation; doi: http://dx.doi.org/10.13039/100001006ADGRL4/ELTD1 is an orphan adhesion GPCR (aGPCR) expressed in endothelial cells that regulates tumour angiogenesis. The majority of aGPCRs are orphan receptors. The Stachel Hypothesis proposes a mechanism for aGPCR activation, in which aGPCRs contain a tethered agonist (termed Stachel) C-terminal to the GPCR-proteolytic site (GPS) cleavage point which, when exposed, initiates canonical GPCR signalling. This has been shown in a growing number of aGPCRs. We tested this hypothesis on ADGRL4/ELTD1 by designing full length (FL) and C-terminal fragment (CTF) ADGRL4/ELTD1 constructs, and a range of potential Stachel peptides. Constructs were transfected into HEK293T cells and HTRF FRET, luciferase-reporter and Alphascreen GPCR signalling assays were performed. A stable ADGRL4/ELTD1 overexpressing HUVEC line was additionally generated and angiogenesis assays, signalling assays and transcriptional profiling were performed. ADGRL4/ELTD1 has the lowest GC content in the aGPCR family and codon optimisation significantly increased its expression. FL and CTF ADGRL4/ELTD1 constructs, as well as Stachel peptides, did not activate canonical GPCR signalling. Furthermore, stable overexpression of ADGRL4/ELTD1 in HUVECs induced sprouting angiogenesis, lowered in vitro anastomoses, and decreased proliferation, without activating canonical GPCR signalling or MAPK/ERK, PI3K/AKT, JNK, JAK/HIF-1α, beta catenin or STAT3 pathways. Overexpression upregulated ANTXR1, SLC39A6, HBB, CHRNA, ELMOD1, JAG1 and downregulated DLL4, KIT, CCL15, CYP26B1. ADGRL4/ELTD1 specifically regulates the endothelial tip-cell phenotype through yet undefined signalling pathways
protocol of a prospective, longitudinal study
Background Natural killer (NK) cells comprise the main components of
lymphocyte-mediated nonspecific immunity. Through their effector function they
play a crucial role combating bacterial and viral challenges. They are also
thought to be key contributors to the systemic spinal cord injury-induced
immune-deficiency syndrome (SCI-IDS). SCI-IDS increases susceptibility to
infection and extends to the post-acute and chronic phases after SCI. Methods
and design The prospective study of NK cell function after traumatic SCI was
carried out in two centers in Berlin, Germany. SCI patients and control
patients with neurologically silent vertebral fracture also undergoing
surgical stabilization were enrolled. Furthermore healthy controls were
included to provide reference data. The NK cell function was assessed at 7
(5â9) days, 14 days (11â28) days, and 10 (8â12) weeks post-trauma. Clinical
documentation included the American Spinal Injury Association (ASIA)
impairment scale (AIS), neurological level of injury, infection status,
concomitant injury, and medications. The primary endpoint of the study is
CD107a expression by NK cells (cytotoxicity marker) 8â12 weeks following SCI.
Secondary endpoints are the NK cellâs TNF-α and IFN-Îł production by the NK
cells 8â12 weeks following SCI. Discussion The protocol of this study was
developed to investigate the hypotheses whether i) SCI impairs NK cell
function throughout the post-acute and sub-acute phases after SCI and ii) the
degree of impairment relates to lesion height and severity. A deeper
understanding of the SCI-IDS is crucial to enable strategies for prevention of
infections, which are associated with poor neurological outcome and elevated
mortality. Trial registration DRKS00009855
The expanding functional roles and signaling mechanisms of adhesion G proteinâcoupled receptors
The adhesion class of G proteinâcoupled receptors (GPCRs) is the second largest family of GPCRs (33 members in humans). Adhesion GPCRs (aGPCRs) are defined by a large extracellular Nâterminal region that is linked to a Câterminal seven transmembrane (7TM) domain via a GPCRâautoproteolysis inducing (GAIN) domain containing a GPCR proteolytic site (GPS). Most aGPCRs undergo autoproteolysis at the GPS motif, but the cleaved fragments stay closely associated, with the Nâterminal fragment (NTF) bound to the 7TM of the Câterminal fragment (CTF). The NTFs of most aGPCRs contain domains known to be involved in cellâcell adhesion, while the CTFs are involved in classical G protein signaling, as well as other intracellular signaling. In this workshop report, we review the most recent findings on the biology, signaling mechanisms, and physiological functions of aGPCRs
The Adhesion G Protein-Coupled Receptor GPR97/ADGRG3 Is Expressed in Human Granulocytes and Triggers Antimicrobial Effector Functions
The adhesion family of G protein-coupled receptors (aGPCRs) comprises 33 members in human, several of which are distinctly expressed and functionally involved in polymorphonuclear cells (PMNs). As former work indicated the possible presence of the aGPCR GPR97 in granulocytes, we studied its cellular distribution, molecular structure, signal transduction, and biological function in PMNs. RNA sequencing and mass-spectrometry revealed abundant RNA and protein expression of ADGRG3/GPR97 in granulocyte precursors and terminally differentiated neutrophilic, eosinophilic, and basophilic granulocytes. Using a newly generated GPR97-specific monoclonal antibody, we confirmed that endogenous GPR97 is a proteolytically processed, dichotomous, N-glycosylated receptor. GPR97 was detected in tissue-infiltrating PMNs and upregulated during systemic inflammation. Antibody ligation of GPR97 increased neutrophil reactive oxygen species production and proteolytic enzyme activity, which is accompanied by an increase in mitogen-activated protein kinases and IÎșBα phosphorylation. In-depth analysis of the GPR97 signaling cascade revealed a possible switch from basal Gαs/cAMP-mediated signal transduction to a Gαi-induced reduction in cAMP levels upon mutation-induced activation of the receptor, in combination with an increase in downstream effectors of GÎČÎł, such as SRE and NF-ÎșB. Finally, ligation of GPR97 increased the bacteria uptake and killing activity of neutrophils. We conclude that the specific presence of GPR97 regulates antimicrobial activity in human granulocytes
Structural and functional evolution of the P2Y12-like receptor group
Metabotropic pyrimidine and purine nucleotide receptors (P2Y receptors) belong to the superfamily of G protein-coupled receptors (GPCR). They are distinguishable from adenosine receptors (P1) as they bind adenine and/or uracil nucleotide triphosphates or diphosphates depending on the subtype. Over the past decade, P2Y receptors have been cloned from a variety of tissues and species, and as many as eight functional subtypes have been characterized. Most recently, several members of the P2Y12-like receptor group, which includes the clopidogrel-sensitive ADP receptor P2Y12, have been deorphanized. The P2Y12-like receptor group comprises several structurally related GPCR which, however, display heterogeneous agonist specificity including nucleotides, their derivatives, and lipids. Besides the established function of P2Y12 in platelet activation, expression in macrophages, neuronal and glial cells as well as recent results from functional studies implicate that several members of this group may have specific functions in neurotransmission, inflammation, chemotaxis, and response to tissue injury. This review focuses specifically on the structure-function relation and shortly summarizes some aspects of the physiological relevance of P2Y12-like receptor members