Activation of the adhesion GPCR GPR133 (ADGRD1) by antibodies targeting the N-terminus

We recently demonstrated that GPR133 (ADGRD1), an adhesion G protein-coupled receptor (aGPCR) whose canonical signaling raises cytosolic cAMP, is necessary for growth of glioblastoma (GBM) and is de novo expressed in GBM relative to normal brain tissue. We showed that dissociation of autoproteolytically generated N-terminal and C-terminal fragments (NTF and CTF) of GPR133 at the plasma membrane promotes receptor activation and increases signaling. Toward developing biologics modulating GPR133 function, we tested antibodies against the N-terminus of GPR133 for effects on receptor signaling. Treatment of HEK293T cells overexpressing GPR133 with such antibodies increased cAMP levels in a concentration-dependent manner. Analysis of supernatants following antibody treatment revealed complexes of the antibodies with the autoproteolytically cleaved NTF of GPR133. Cells expressing a cleavage-deficient mutant GPR133 (H543R) did not respond to antibody stimulation, suggesting that the effect is cleavage-dependent. The antibody-mediated stimulation of wild-type GPR133, but not the cleavage-deficient H543R mutant, was reproducible in patient-derived GBM cells. These findings provide a paradigm for modulation of GPR133 function with biologics and support the hypothesis that NTF-CTF dissociation promotes receptor activation and signaling.

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Dissociation of the intramolecularly cleaved N- and C-terminal fragments of the adhesion G protein-coupled receptor GPR133 (ADGRD1) increases canonical signaling

10.1101/2020.12.08.415398 ◽

2020 ◽

Author(s):

Joshua D. Frenster ◽

Gabriele Stephan ◽

Niklas Ravn-Boess ◽

Devin Bready ◽

Jordan Wilcox ◽

...

Keyword(s):

Plasma Membrane ◽

G Protein ◽

Receptor Activation ◽

List Type ◽

G Protein Coupled Receptor ◽

Major Mechanism ◽

N Terminus ◽

Adhesion Gpcr ◽

G Protein Coupled

SUMMARYGPR133 (ADGRD1), an adhesion G protein-coupled receptor (GPCR), is necessary for growth of glioblastoma (GBM), a brain malignancy. The extracellular N-terminus of GPR133 is thought to be autoproteolytically cleaved into an N-terminal and a C-terminal fragment (NTF and CTF). Nevertheless, the role of this cleavage in receptor activation remains unclear. Here, we show that the wild-type (WT) receptor is cleaved after protein synthesis and generates significantly more canonical signaling than an uncleavable point mutant (H543R) in patient-derived GBM cultures and HEK293T cells. However, the resulting NTF and CTF remain non-covalently bound until the receptor is trafficked to the plasma membrane, where we find NTF-CTF dissociation. Using a fusion of the hPAR1 receptor N-terminus and the CTF of GPR133, we demonstrate that thrombin-induced cleavage and shedding of the hPAR1 NTF increases receptor signaling. This study supports a model where dissociation of the NTF at the plasma membrane promotes GPR133 activation.Highlights-GPR133 is intramolecularly cleaved in patient-derived GBM cultures-Cleaved GPR133 signals at higher efficacy than the uncleavable GPR133 H543R mutant-The N- and C-terminal fragments (NTF and CTF) of GPR133 dissociate at the plasma membrane-Acute thrombin-induced cleavage of the human PAR1 NTF from the GPR133 CTF increases signalingeTOC BlurbFrenster et al. demonstrate intramolecular cleavage of the adhesion GPCR GPR133 in glioblastoma and HEK293T cells. The resulting N- and C-terminal fragments dissociate at the plasma membrane to increase canonical signaling. The findings suggest dissociation of GPR133’s N-terminus at the plasma membrane represents a major mechanism of receptor activation.

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CSIG-21. DE-ORPHANIZING GPR133 - AN ADHESION GPCR REQUIRED FOR GLIOBLASTOMA PROGRESSION

Neuro-Oncology ◽

10.1093/neuonc/noz175.191 ◽

2019 ◽

Vol 21 (Supplement_6) ◽

pp. vi48-vi48

Author(s):

Joshua Frenster ◽

Hediye Erdjument-Bromage ◽

Gabriele Stephan ◽

Shravanthi Chidambaram ◽

Abdulhakeem Alghamdi ◽

...

Keyword(s):

Extracellular Matrix ◽

Ligand Binding ◽

De Novo ◽

Receptor Activation ◽

Extracellular Proteins ◽

Hek293 Cells ◽

Migration And Invasion ◽

Tumor Cell Migration ◽

Adhesion Gpcr ◽

Camp Levels

Abstract We previously found GPR133 (ADGRD1), an orphan adhesion GPCR, is de novo expressed in glioblastoma (GBM) and enriched in patient-derived glioblastoma stem cells (GSCs). Knockdown of GPR133 reduces GBM cell proliferation and tumorsphere formation, and abolishes orthotopic tumor initiation in vivo in mice. Analysis of TCGA data indicates that increased GPR133 transcription inversely correlates with patient survival in GBM. While these findings underscore the importance of GPR133 in GBM and suggest an essential role in tumor growth, its ligand and mechanism of activation remain unknown. Toward identifying GPR133 ligands, we used GPR133’s N-terminal ectodomain as bait and performed affinity co-immunoprecipitation (CoIP) followed by mass spectrometry as an unbiased screening approach. We identified 490 extracellular proteins with enriched binding to GPR133 compared to control. Reverse CoIP using the 15 most abundant candidate ligands as bait to purify the receptor confirmed this interaction reproducibly in 4 candidates. Despite this binding, overexpression of these candidate ligands, or addition of purified recombinant protein, is not sufficient to increase receptor signaling as assessed by cAMP levels in HEK293 cells. This suggests that ligand binding to the GPR133 ectodomain may not be sufficient by itself to induce receptor activation. We hypothesize receptor activation requires mechanical forces in addition to ligand binding. Consistent with this hypothesis, the GPR133 binding proteins we have identified may be anchored to the extracellular matrix, mediating such mechanical force. To test whether mechanical shearing of the extracellular domain is sufficient for receptor activation, we used Dynabeads coupled to antibody against GPR133’s N-terminal ectodomain, and indeed observed receptor activation leading to elevated cAMP levels. No activation was observed when Dynabeads devoid of antibody were used. This mode of GPR133 activation might indicate a role in sensing mechanical/viscoelastic properties of GBM extracellular matrix, which may be relevant to tumor cell migration and invasion.

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CSIG-07. ACTIVATION OF THE ADHESION G PROTEIN-COUPLED RECEPTOR GPR133 BY ANTIBODIES AGAINST THE N-TERMINUS

Neuro-Oncology ◽

10.1093/neuonc/noab196.133 ◽

2021 ◽

Vol 23 (Supplement_6) ◽

pp. vi34-vi34

Author(s):

Gabriele Stephan ◽

Joshua Frenster ◽

Niklas Ravn-Boess ◽

Devin Bready ◽

Jordan Wilcox ◽

...

Keyword(s):

Plasma Membrane ◽

G Protein ◽

Receptor Activation ◽

Cell Culture Supernatant ◽

Dependent Manner ◽

G Protein Coupled Receptor ◽

Terminal Fragment ◽

C Terminus ◽

N Terminus ◽

G Protein Coupled

Abstract We recently demonstrated that GPR133 (ADGRD1), a member of the adhesion G protein-coupled receptor (aGPCR) family, is necessary for growth of glioblastoma (GBM) and is de novo expressed in GBM relative to normal brain tissue. We therefore postulate that GPR133 represents a novel target in GBM, which merits development of therapeutics. Like most aGPCRs, GPR133 is characterized by an intracellular C-terminus, 7 plasma membrane-spanning α-helices and a large extracellular N-terminus. The N-terminus possesses a conserved GPCR autoproteolysis-inducing (GAIN) domain that catalyzes cleavage at a GPCR proteolysis site (GPS), resulting in a C-terminal fragment (CTF) and an N-terminal fragment (NTF). We showed that dissociation of the cleaved NTF and CTF at the plasma membrane increases canonical signaling of GPR133, which is mediated by coupling to Gs and increase in cytosolic cAMP. Toward characterizing the effect of biologics on GPR133 function, we overexpressed wild-type or mutant forms of GPR133 in HEK293T cells and patient-derived GBM cells lines. Treatment of these cells with antibodies specifically targeting the NTF of GPR133 increased receptor activation in a dose-dependent manner. No effects were elicited with an antibody against the receptor’s intracellular C-terminus. Interestingly, cells overexpressing a cleavage-deficient mutant GPR133 (H543R) did not respond to antibody stimulation, suggesting that the effect is cleavage-dependent. Following antibody treatment, co-purification of the GPR133 NTF and the N-terminal antibody from the cell culture supernatant indicated the formation of antibody-NTF complexes. Analysis of these complexes suggested that antibody binding stimulated the dissociation of the NTF from the CTF. However, the increased flexibility of the GAIN domain and NTF after cleavage, independently of dissociation, may also endow the receptor with responsiveness to the effects of the antibodies. These data constitute a proof-of-concept paradigm of modulation of GPR133 function with antibodies. This work provides rationale for pursuing development of biologics targeting GPR133 in GBM.

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Calcium-sensitivity of inositol 1,4,5-trisphosphate metabolism in exocrine cells from the avian salt gland

Biochemical Journal ◽

10.1042/bj2820703 ◽

1992 ◽

Vol 282 (3) ◽

pp. 703-710 ◽

Cited By ~ 15

Author(s):

J P Hildebrandt ◽

T J Shuttleworth

Keyword(s):

Substrate Concentration ◽

Inositol Phosphates ◽

Receptor Activation ◽

Dependent Manner ◽

Salt Glands ◽

Concentration Dependent Manner ◽

Intact Cells ◽

Exocrine Cells ◽

Tissue Homogenates

The generation of inositol phosphates upon muscarinic-receptor activation was studied in [3H]inositol-loaded exocrine cells from the nasal salt glands of the duck Anas platyrhynchos, and the metabolism of different inositol phosphates in vitro was studied in tissue homogenates, with particular reference to the possible interaction of changes in intracellular [Ca2+] ([Ca2+]i) with the metabolic processes. In intact cells, there was a rapid (within 15 s) generation of Ins(1,4,5)P3 and Ins(1,3,4,5)P4, followed by an accumulation of their breakdown products, Ins(1,3,4)P3 and inositol bis- and monophosphates. Ca(2+)-sensitivity of the Ins(1,4,5)P3 3-kinase was demonstrated in tissue homogenates, with the rate of phosphorylation increasing 2-fold at free Ca2+ concentrations greater than 1 microM. However, addition of calmodulin or the presence of the calmodulin inhibitor W-7 (up to 100 microM) had no effect. 3-Kinase activity increased proportionally with the initial Ins(1,4,5)P3 concentration up to 1 microM, but a 10-fold higher substrate concentration produced only a doubling in the phosphorylation rate. Ins(1,3,4,5)P4 was dephosphorylated to Ins(1,3,4)P3, which accumulated in the homogenate assays as well as in intact cells. Depending on its concentration, Ins(1,3,4)P3 was phosphorylated [in part to Ins(1,3,4,6)P4] or dephosphorylated. To investigate the Ca(2+)-sensitivity of the 3-kinase in intact cells, excess quin2 was used to buffer the receptor-mediated transient changes in [Ca2+]i in [3H]inositol-loaded cells. These experiments revealed that increasing [Ca2+]i from less than 100 to approx. 400 nM (i.e. within the physiological range) has no effect on the partitioning of Ins(1,4,5)P3 metabolism (phosphorylation versus dephosphorylation) and on the accumulation of Ins(1,4,5)P3 and Ins(1,3,4,5)P4. This indicates that activation of the 3-kinase by physiologically relevant Ca2+ concentrations may not play a major role in the generation of Ins(1,3,4,5)P4 signals upon receptor activation in these cells. The latter are mainly achieved by the receptor-mediated increase in Ins(1,4,5)P3 in the cell and its phosphorylation by the 3-kinase in a substrate-concentration-dependent manner.

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Regulation of Na(+)-3HCO3- cotransport in rabbit proximal convoluted tubule via adenosine A1 receptor

AJP Renal Physiology ◽

10.1152/ajprenal.1993.265.4.f511 ◽

1993 ◽

Vol 265 (4) ◽

pp. F511-F519 ◽

Cited By ~ 14

Author(s):

M. Takeda ◽

K. Yoshitomi ◽

M. Imai

Keyword(s):

Basolateral Membrane ◽

Receptor Activation ◽

Proximal Convoluted Tubule ◽

Adenosine A1 Receptor ◽

Intracellular Camp ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Adenosine A1 ◽

A1 Receptor

We investigated the role of adenosine A1-receptor in the regulation of basolateral Na(+)-3HCO3- cotransporter in the rabbit proximal convoluted tubule (PCT) microperfused in vitro by monitoring basolateral membrane potential and intracellular pH. FK-453, a highly specific A1 antagonist, inhibited basolateral HCO3- conductance in a concentration-dependent manner (10(-10)-10(-5) M). Other A1 antagonists, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) at 10(-5) M and theophylline at 10(-3) M, also had similar effects. N6-cyclohexyladenosine (CHA) at 10(-7) M attenuated the effect of low concentration (10(-8) M) of FK-453. Either enhancement of the degradation of adenosine by 0.1 U/ml adenosine deaminase (ADA) or inhibition of adenosine release from the cells by 10(-6) M S-(4-nitrobenzyl)-6-thioinosine (NBTI) mimicked the effects of A1 antagonists. These observations suggest that endogenous adenosine is released from PCT cells and stimulates Na(+)-3HCO3- cotransporter. Both 10(-4) M 8-(4-chlorophenylthio)-adenosine 3',5'-cyclic monophosphate (CPT-cAMP) and 10(-6) M forskolin also inhibited basolateral HCO3- conductance. Both 10(-6) M FK-453 and 10(-4) M CPT-cAMP decreased the initial rate as well as the magnitude of intracellular acidification induced by reduction of peritubular HCO3- concentration from 25 to 0 mM. Neither 10(-6) M FK-453 nor 10(-7) M CHA changed intracellular Ca2+ concentration as measured by fura-2 fluorescence. These results indicate that adenosine might stimulate HCO3- exit across the basolateral membrane through Na(+)-3HCO3- cotransporter by decreasing intracellular cAMP via A1-receptor activation.(ABSTRACT TRUNCATED AT 250 WORDS)

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Influence of ammonia concentration on [15N]ammonia uptake andde novosynthesis of different amino acids by mixed rumen microorganisms from the sheep rumenin vitro

Proceedings of the British Society of Animal Science ◽

10.1017/s1752756200003677 ◽

1999 ◽

Vol 1999 ◽

pp. 212-212 ◽

Cited By ~ 1

Author(s):

C. Atasoglu ◽

C.J. Newbold ◽

R.J. Wallace

Keyword(s):

Amino Acids ◽

Protein Synthesis ◽

De Novo ◽

Ammonia Concentration ◽

Dependent Manner ◽

Microbial Protein ◽

Concentration Dependent Manner ◽

Rumen Microorganisms ◽

Ammonia Uptake

Ammonia is thought to be the main source of nitrogen for protein synthesis by the rumen microorganisms, but peptides and amino acids derived from protein degradation are also incorporated into microbial protein. Recent experiments carried out by Atasogluet al.(1998) demonstrated that preformed amino acids decrease the uptake of ammonia into microbial protein and microbial amino acids in a concentration-dependent manner. However, little is known about how rumen ammonia concentrations affect ammonia uptake into microbial protein. The present study was undertaken to determine the influence of rumen ammonia concentrations on ammonia incorporation andde novosynthesis of individual amino acids by the mixed rumen microorganismsin vitro.

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Presynaptic GABAB Receptors Regulate Retinohypothalamic Tract Synaptic Transmission by Inhibiting Voltage-Gated Ca2+ Channels

Journal of Neurophysiology ◽

10.1152/jn.00909.2005 ◽

2006 ◽

Vol 95 (6) ◽

pp. 3727-3741 ◽

Cited By ~ 25

Author(s):

Mykhaylo G. Moldavan ◽

Robert P. Irwin ◽

Charles N. Allen

Keyword(s):

Glutamate Release ◽

Receptor Activation ◽

Gabab Receptors ◽

Channel Blockers ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Epsc Amplitude ◽

Receptor Modulation ◽

Joint Application ◽

Retinohypothalamic Tract

Presynaptic GABAB receptor activation inhibits glutamate release from retinohypothalamic tract (RHT) terminals in the suprachiasmatic nucleus (SCN). Voltage-clamp whole cell recordings from rat SCN neurons and optical recordings of Ca2+-sensitive fluorescent probes within RHT terminals were used to examine GABAB-receptor modulation of RHT transmission. Baclofen inhibited evoked excitatory postsynaptic currents (EPSCs) in a concentration-dependent manner equally during the day and night. Blockers of N-, P/Q-, T-, and R-type voltage-dependent Ca2+ channels, but not L-type, reduced the EPSC amplitude by 66, 36, 32, and 18% of control, respectively. Joint application of multiple Ca2+ channel blockers inhibited the EPSCs less than that predicted, consistent with a model in which multiple Ca2+ channels overlap in the regulation of transmitter release. Presynaptic inhibition of EPSCs by baclofen was occluded by ω-conotoxin GVIA (≤72%), mibefradil (≤52%), and ω-agatoxin TK (≤15%), but not by SNX-482 or nimodipine. Baclofen reduced both evoked presynaptic Ca2+ influx and resting Ca2+ concentration in RHT terminals. Tertiapin did not alter the evoked EPSC and baclofen-induced inhibition, indicating that baclofen does not inhibit glutamate release by activation of Kir3 channels. Neither Ba2+ nor high extracellular K+ modified the baclofen-induced inhibition. 4-Aminopyridine (4-AP) significantly increased the EPSC amplitude and the charge transfer, and dramatically reduced the baclofen effect. These data indicate that baclofen inhibits glutamate release from RHT terminals by blocking N-, T-, and P/Q-type Ca2+ channels, and possibly by activation of 4-AP–sensitive K+ channels, but not by inhibition of R- and L-type Ca2+ channels or by Kir3 channel activation.

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Effects of estrogen on cerebral blood flow and pial microvasculature in rabbits

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.2000.279.3.h1208 ◽

2000 ◽

Vol 279 (3) ◽

pp. H1208-H1214 ◽

Cited By ~ 20

Author(s):

M. T. Littleton-Kearney ◽

D. M. Agnew ◽

R. J. Traystman ◽

P. D. Hurn

Keyword(s):

Blood Flow ◽

Estrogen Receptor ◽

Cerebral Blood Flow ◽

Receptor Activation ◽

Receptor Subtypes ◽

Dependent Manner ◽

Pial Arteries ◽

Concentration Dependent Manner ◽

Cranial Window ◽

Pial Arterioles

We tested the hypothesis that intracarotid estrogen infusion increases cerebral blood flow (CBF) in a concentration-dependent manner and direct application of estrogen on pial arterioles yields estrogen receptor-mediated vasodilation. Rabbits of both genders were infused with estrogen via a branch of the carotid artery. Estrogen doses of 20 or 0.05 μg · ml−1 · min−1 were used to achieve supraphysiological or physiological plasma estrogen levels, respectively. CBF and cerebral vascular resistance were determined at baseline, during the infusion, and 60-min postinfusion, and effects on pial diameter were assessed via a cranial window. Pial arteriolar response to estrogen alone and to estrogen after administration of tamoxifen (10−7), an antiestrogen drug that binds to both known estrogen receptor subtypes, was tested. No gender differences were observed; therefore, data were combined for both males and females. Systemic estrogen infusion did not increase regional CBF. Estradiol dilated pial arteries only at concentrations ranging from 10−4–10−7 M ( P ≤ 0.05). Pretreatment with tamoxifen alone had no effect on arteriolar diameter but inhibited estrogen-induced vasodilation ( P < 0.001). Our data suggest that estrogen does not increase CBF under steady-state conditions in rabbits. In the pial circulation, topically applied estradiol at micromolar concentrations dilates vessels. The onset is rapid and dependent on estrogen receptor activation.

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Inhibitory action of somatostatin-14 on hormone-stimulated cyclic adenosine monophosphate induction in porcine granulosa and luteal cells

Journal of Endocrinology ◽

10.1677/joe.0.1340297 ◽

1992 ◽

Vol 134 (2) ◽

pp. 297-306 ◽

Cited By ~ 12

Author(s):

K. Rajkumar ◽

D. E. Kerr ◽

R. N. Kirkwood ◽

B. Laarveld

Keyword(s):

Cyclic Adenosine Monophosphate ◽

Adenosine Monophosphate ◽

Inhibitory Effect ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Luteal Cells ◽

Camp Generation ◽

Camp Induction ◽

Camp Levels ◽

Somatostatin 14

ABSTRACT Somatostatin-14 (SRIF-14) inhibited, in a concentration-dependent manner, LH- and forskolin-stimulated cyclic adenosine monophosphate (cAMP) induction in porcine granulosa and luteal cells. The inhibitory effect of SRIF-14 on hormone-induced cAMP generation was more potent in porcine ovarian cells than in the GH-3 pituitary cell line. The inhibitory effect of SRIF-14 was impeded by neutralizing its biological activity with specific antiserum. Preincubation of luteal and granulosa cells with phorbol 12-myristate 13-acetate (PMA) enhanced LH- and forskolin-stimulated cAMP levels. SRIF-14 failed to inhibit LH- or forskolin-stimulated cAMP levels in cells preincubated with PMA. It is concluded that SRIF-14 inhibits hormone-stimulated cAMP induction in the porcine ovary. LH-induced protein kinase C activation may be physiologically important to alleviate the inhibitory effects of SRIF-14. Journal of Endocrinology (1992) 134, 297–306

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Mapping the Protease Activated Receptor 4 (PAR4) Homodimer Interface.

Blood ◽

10.1182/blood.v114.22.773.773 ◽

2009 ◽

Vol 114 (22) ◽

pp. 773-773

Author(s):

Marvin T Nieman

Keyword(s):

Conflicts Of Interest ◽

Specific Interaction ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Receptor Activation ◽

Dependent Manner ◽

Intracellular Loop ◽

Concentration Dependent Manner ◽

Platelet Surface

Abstract Abstract 773 Thrombin activates platelets by binding and cleaving protease activated receptors 1 and 4 (PAR1 and PAR4). PAR1 and PAR4 communicate with each other to lower the concentration of thrombin required for PAR4 activation (Nieman Biochemistry, 2008). In addition, PAR1 and PAR4 form homo and heterodimers. However, where these receptors interact has not been defined and it is not known if dimerization influences receptor activation, downstream signaling, or both. Since PAR4 activation is important on human and mouse platelets, we sought to characterize the interaction site between PAR4 homodimers. Using bioluminescence resonance energy transfer (BRET), we mapped the PAR4 homodimer interface. The PAR4 homodimers show a specific interaction as indicated by a hyperbolic BRET signal in response to increasing PAR4-GFP expression with a fixed concentration of PAR4-Rluc. The threshold maximum BRET signal was disrupted in a concentration-dependent manner by unlabeled PAR4. In contrast, the unrelated G-protein coupled receptor, rhodopsin, was unable to disrupt the BRET signal indicating that the disruption of the PAR4 homodimer is a specific interaction. We have mapped the region required for PAR4 homodimer formation using chimeras between rhodopsin and PAR4. PAR4 does not interact with rhodopsin in BRET assays. Using a library of rho-PAR4 chimeras that have the junction at the beginning of transmembrane (TM) 2, 3, 4, 5, 6 or 7, we determined where dimer formation is restored. When the junction is placed at the beginning of TM4 or TM5, the chimera does not interact with PAR4-WT. In contrast, when the junction is moved to the end of TM2, the BRET signal is restored. These results indicate that the region on PAR4 required for homodimer formation encompasses a 63 amino acid region that includes the first extracellular loop, TM3 and the second intracellular loop. These studies establish techniques that may be used to define the interactions between other GPCRs found on the platelet surface. These receptor-receptor interactions may be another level of regulation of agonist activity and platelet function in vivo and may provide novel targets for anti-platelet therapies. Disclosures: No relevant conflicts of interest to declare.

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