Functional impact of intramolecular cleavage and dissociation of adhesion G protein–coupled receptor GPR133 (ADGRD1) on canonical signaling

Abstract

GPR133 (ADGRD1), an adhesion G protein–coupled receptor (GPCR) whose canonical signaling activates GαS-mediated generation of cytosolic cAMP, has been shown to be necessary for the growth of glioblastoma (GBM), a brain malignancy. The extracellular N terminus of GPR133 is thought to be autoproteolytically cleaved into N-terminal and C- terminal fragments (NTF and CTF, respectively). However, the role of this cleavage in receptor activation remains unclear. Here, we used subcellular fractionation and immunoprecipitation approaches to show that the WT GPR133 receptor is cleaved shortly after protein synthesis and generates significantly more canonical signaling than an uncleavable point mutant GPR133 (H543R) in patient-derived GBM cultures and HEK293T cells. After cleavage, the resulting NTF and CTF remain noncovalently bound to each other until the receptor is trafficked to the plasma membrane, where we demonstrated NTF–CTF dissociation occurs. Using a fusion of the CTF of GPR133 and the N terminus of thrombin-activated human protease-activated receptor 1 as a controllable proxy system to test the effect of intramolecular cleavage and dissociation, we also showed that thrombin-induced cleavage and shedding of the human protease-activated receptor 1 NTF increased intracellular cAMP levels. These results support a model wherein dissociation of the NTF from the CTF at the plasma membrane promotes GPR133 activation and downstream signaling. These findings add depth to our understanding of the molecular life cycle and mechanism of action of GPR133 and provide critical insights that will inform therapeutic targeting of GPR133 in GBM. GPR133 (ADGRD1), an adhesion G protein–coupled receptor (GPCR) whose canonical signaling activates GαS-mediated generation of cytosolic cAMP, has been shown to be necessary for the growth of glioblastoma (GBM), a brain malignancy. The extracellular N terminus of GPR133 is thought to be autoproteolytically cleaved into N-terminal and C- terminal fragments (NTF and CTF, respectively). However, the role of this cleavage in receptor activation remains unclear. Here, we used subcellular fractionation and immunoprecipitation approaches to show that the WT GPR133 receptor is cleaved shortly after protein synthesis and generates significantly more canonical signaling than an uncleavable point mutant GPR133 (H543R) in patient-derived GBM cultures and HEK293T cells. After cleavage, the resulting NTF and CTF remain noncovalently bound to each other until the receptor is trafficked to the plasma membrane, where we demonstrated NTF–CTF dissociation occurs. Using a fusion of the CTF of GPR133 and the N terminus of thrombin-activated human protease-activated receptor 1 as a controllable proxy system to test the effect of intramolecular cleavage and dissociation, we also showed that thrombin-induced cleavage and shedding of the human protease-activated receptor 1 NTF increased intracellular cAMP levels. These results support a model wherein dissociation of the NTF from the CTF at the plasma membrane promotes GPR133 activation and downstream signaling. These findings add depth to our understanding of the molecular life cycle and mechanism of action of GPR133 and provide critical insights that will inform therapeutic targeting of GPR133 in GBM. The adhesion family of G protein–coupled receptors (GPCRs) has attracted increasing interest in the recent years for essential functions in health and disease (1Morgan R.K. Anderson G.R. Arac D. Aust G. Balenga N. Boucard A. Bridges J.P. Engel F.B. Formstone C.J. Glitsch M.D. Gray R.S. Hall R.A. Hsiao C.C. Kim H.Y. Knierim A.B. et al.The expanding functional roles and signaling mechanisms of adhesion G protein-coupled receptors.Ann. N. Y. Acad. Sci. 2019; 1456: 5-25Crossref PubMed Scopus (5) Google Scholar, 2Langenhan T. Adhesion G protein-coupled receptors-candidate metabotropic mechanosensors and novel drug targets.Basic Clin. Pharmacol. Toxicol. 2019; 126 Suppl 6: 5-16PubMed Google Scholar). The large extracellular N termini of adhesion GPCRs contain a GPCR autoproteolysis-inducing domain, which is thought to catalyze autoproteolytic cleavage at the GPCR proteolysis site (GPS) marked by the tripeptide sequence H-L/I-∗-S/T (∗ denotes the cleavage site) (3Lin H.H. Stacey M. Yona S. Chang G.W. GPS proteolytic cleavage of adhesion-GPCRs.Adv. Exp. Med. Biol. 2010; 706: 49-58Crossref PubMed Scopus (24) Google Scholar, 4Arac D. Boucard A.A. Bolliger M.F. Nguyen J. Soltis S.M. Sudhof T.C. Brunger A.T. A novel evolutionarily conserved domain of cell-adhesion GPCRs mediates autoproteolysis.EMBO J. 2012; 31: 1364-1378Crossref PubMed Scopus (226) Google Scholar). After this intramolecular cleavage, adhesion GPCRs are generally believed to exist as noncovalently bound heterodimers of their extracellular N-terminal fragment (NTF) and transmembrane-spanning C-terminal fragment (CTF) (5Gray J.X. Haino M. Roth M.J. Maguire J.E. Jensen P.N. Yarme A. Stetler-Stevenson M.A. Siebenlist U. Kelly K. CD97 is a processed, seven-transmembrane, heterodimeric receptor associated with inflammation.J. Immunol. 1996; 157: 5438-5447PubMed Google Scholar, 6Krasnoperov V. Bittner M.A. Holz R.W. Chepurny O. Petrenko A.G. Structural requirements for alpha-latrotoxin binding and alpha-latrotoxin-stimulated secretion. A study with calcium-independent receptor of alpha-latrotoxin (CIRL) deletion mutants.J. Biol. Chem. 1999; 274: 3590-3596Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). The recent demonstration of a also as the C-terminal to the has to the that NTF–CTF dissociation the for the sequence to receptor activation J. N. A. M. S. T. A the activates the adhesion G protein-coupled receptors and Full Text Full Text PDF PubMed Scopus Google Scholar). However, the mechanism of receptor activation by autoproteolytic cleavage and NTF–CTF dissociation is to of the adhesion GPCR adhesion GPCRs been to intramolecular cleavage, and cleavage is for their T. Adhesion G protein-coupled receptors-candidate metabotropic mechanosensors and novel drug targets.Basic Clin. Pharmacol. Toxicol. 2019; 126 Suppl 6: 5-16PubMed Google Scholar, V. J. S. T. The of the adhesion GPCR is by J. PubMed Scopus Google Scholar, M. M. mediates by to Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, S. M. S. D. A. T. T. The GPS is a molecular for of adhesion G protein-coupled 2012; Full Text Full Text PDF PubMed Scopus Google Scholar, J. T. adhesion receptor GPR133 to Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). in adhesion cleavage in D. Boucard A.A. Bolliger M.F. Nguyen J. Soltis S.M. Sudhof T.C. Brunger A.T. A novel evolutionarily conserved domain of cell-adhesion GPCRs mediates autoproteolysis.EMBO J. 2012; 31: 1364-1378Crossref PubMed Scopus (226) Google Scholar, T. K. K. K. N. G protein-coupled receptor a G and Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, C.C. H.Y. Stacey M. Chang G.W. H.H. the GPS of PubMed Scopus Google Scholar, Y. M. M.J. S.M. H.Y. of the adhesion G protein-coupled receptor CD97 to and Pharmacol. PubMed Scopus Google Scholar). and signaling been for adhesion GPCRs V. J. S. T. The of the adhesion GPCR is by J. PubMed Scopus Google Scholar, S. M. S. D. A. T. T. The GPS is a molecular for of adhesion G protein-coupled 2012; Full Text Full Text PDF PubMed Scopus Google Scholar, A. Hall R.A. and signaling by the adhesion G protein-coupled receptors and Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, S. A. A. S. Arac D. of signaling by to extracellular Acad. Sci. U. S. A. PubMed Scopus Google Scholar, M.J. S. A. T. A. Engel F.B. of adhesion G protein-coupled receptor is Acad. Sci. U. S. A. PubMed Scopus Google Scholar). These the to study mechanisms of activation for adhesion GPCRs an and in that GPR133 (ADGRD1), a of the adhesion family of is and for growth glioblastoma (GBM), an brain J. K. D. M. D. et (ADGRD1), an adhesion is necessary for glioblastoma PubMed Scopus Google Scholar, M. S. J. J. D. D. N. G. D. et of the adhesion G protein-coupled receptor GPR133 in Google Scholar, J. A. D. GPR133 promotes glioblastoma growth in PubMed Scopus Google Scholar). CTF of GPR133 significantly more G signaling than the receptor J. N. A. M. S. T. A the activates the adhesion G protein-coupled receptors and Full Text Full Text PDF PubMed Scopus Google Scholar). has been study of the of GPR133 cleavage the NTF–CTF Here, we that GPR133 is cleaved in patient-derived GBM and that cleaved GPR133 has a than an uncleavable GPR133 point the cleaved CTF and NTF remain noncovalently bound to each other the we that the NTF from the CTF the plasma membrane Using a fusion protein of the N terminus from human protease-activated receptor 1 receptor and the CTF of we show that dissociation of the NTF and of the CTF at the canonical signaling of These findings an NTF–CTF dissociation model for activation of GPR133 signaling. canonical signaling by GPR133 is to resulting in an of intracellular cAMP J. T. adhesion receptor GPR133 to Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). that of GPR133 in HEK293T is associated with in cAMP as by a cAMP other GPCR signaling test intramolecular cleavage has for canonical we an mutant GPR133 a point at the of the GPS cleavage site J. T. adhesion receptor GPR133 to Biol. Chem. Full Text Full Text PDF PubMed Scopus Google and and A and is to GPR133 cleavage cAMP signaling J. N. A. M. S. T. A the activates the adhesion G protein-coupled receptors and Full Text Full Text PDF PubMed Scopus Google Scholar, J. T. adhesion receptor GPR133 to Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). used to the cAMP by the cleaved WT and mutant receptor in HEK293T cells. of receptor significantly cAMP a signaling of the uncleavable mutant GPR133 increased cAMP to of the cAMP with WT GPR133 in signaling be by in of the as by has demonstrated that GPR133 is in human GBM M. S. J. J. D. D. N. G. D. et of the adhesion G protein-coupled receptor GPR133 in Google as as our patient-derived GBM where is for growth J. K. D. M. D. et (ADGRD1), an adhesion is necessary for glioblastoma PubMed Scopus Google Scholar, J. A. D. GPR133 promotes glioblastoma growth in PubMed Scopus Google Scholar). to our findings in the of patient-derived GBM cultures and results and These findings that autoproteolytic cleavage receptor the of intramolecular cleavage of we WT GPR133 and the point mutant in HEK293T and by with a the GPR133 CTF and our the GPR133 NTF J. K. D. M. D. et (ADGRD1), an adhesion is necessary for glioblastoma PubMed Scopus Google Scholar, M. S. J. J. D. D. N. G. D. et of the adhesion G protein-coupled receptor GPR133 in Google and for the CTF and the NTF the CTF, the the receptor fragments to CTF and NTF with increased the CTF, and the the WT we also a at which we to the WT as as a a of the CTF and the GPR133 as a with cleavage and the of intramolecular cleavage of adhesion GPCRs has been as D. Boucard A.A. Bolliger M.F. Nguyen J. Soltis S.M. Sudhof T.C. Brunger A.T. A novel evolutionarily conserved domain of cell-adhesion GPCRs mediates autoproteolysis.EMBO J. 2012; 31: 1364-1378Crossref PubMed Scopus (226) Google Scholar, T. K. K. K. N. G protein-coupled receptor a G and Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, C.C. H.Y. Stacey M. Chang G.W. H.H. the GPS of PubMed Scopus Google Scholar, Y. M. M.J. S.M. H.Y. of the adhesion G protein-coupled receptor CD97 to and Pharmacol. PubMed Scopus Google we GPR133 cleavage in our patient-derived GBM the cleavage in patient-derived GBM cultures receptor fragments in and in is to the and molecular of the and The of the receptor the is of the NTF and CTF are to be the and The in the of the receptor and NTF are to as demonstrated in the The in the of the CTF from to is by increased the of the CTF, as for other A. M. G. binding of membrane Acad. Sci. U. S. A. PubMed Scopus Google Scholar). findings that GPR133 is cleaved in human GBM and HEK293T cells. mechanisms the increased signaling by WT GPR133 with the uncleavable mutant we their to the the Using and we the WT and the GPR133 at the of and the WT and the GPR133 demonstrated in intracellular of the as as at the and findings we used subcellular fractionation and to for with and the and the is that the showed for subcellular the as demonstrated by of for by the cleaved NTF and CTF of the WT as as the in the and with our findings that intramolecular cleavage of GPR133 is for subcellular to the and that the in signaling the cleaved and GPR133 is to be by subcellular the of GPR133 the subcellular that the cleaved WT GPR133 NTF and the GPR133 an from in the from the in the and and respectively). are the NTF we that are to of as the receptor the test this we the subcellular with an and the of cleaved WT NTF and GPR133 to the of their subcellular of and and are shown in for that the protein with of WT GPR133 the we a in the from to and the to our that this is the of WT GPR133 we in from and and and The of this results from increased binding the of the receptor A. 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PubMed Scopus Google Scholar). a fusion protein the human N terminus and CTF and to the cleavage of the NTF and dissociation from the showed that this thrombin-induced NTF shedding at the the of canonical signaling of the fusion WT GPR133 the demonstrated that GPR133 is cleaved and that the dissociation of the NTF promotes receptor activation and canonical our will mechanisms NTF–CTF dissociation at the mechanism of dissociation is that binding the the CTF N. T. Adhesion GPCRs as a of metabotropic Exp. Pharmacol. PubMed Scopus Google Scholar, A.G. Adhesion G protein-coupled receptors are by of a Acad. Sci. U. S. A. PubMed Scopus Google Scholar, S. A. M. T. 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M.J. The for protein and PubMed Scopus Google Scholar). The in the the sequence as the of the to be with The resulting in in a and the as a has been and as an in as the test and with The of at are the S. T. A. N. T. A. is by in the adhesion GPCR Chem. Biol. PubMed Scopus Google Scholar, S. M.J. The for protein and PubMed Scopus Google Scholar). D. G. and of a in the for the of GPR133 as a in D. G. has from and G. is an and of the at of for with J. D. and and G. S. and and N. and and D. and and J. N. T. and and and G. and and D. G. and and J. D. by a to of G. S. by a N. by a to of and T. S. by to T. S. and to and D. G. by at of of and as a The by the The is the of the and the of the of

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