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Residue-level determinants of RGS R4 subfamily GAP activity and specificity towards the Gi subfamily

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Abstract

The structural basis for the GTPase-accelerating activity of regulators of G protein signaling (RGS) proteins, as well as the mechanistic basis for their specificity in interacting with the heterotrimeric (αβγ) G proteins they inactivate, is not sufficiently understood at the family level. Here, we used biochemical assays to compare RGS domains across the RGS family and map those individual residues that favorably contribute to GTPase-accelerating activity, and those residues responsible for attenuating RGS domain interactions with Gα subunits. We show that conserved interactions of RGS residues with both the Gα switch I and II regions are crucial for RGS activity, while the reciprocal effects of “modulatory” and “disruptor” residues selectively modulate RGS activity. Our results quantify how specific interactions between RGS domains and Gα subunits are set by a balance between favorable RGS residue interactions with particular Gα switch regions, and unfavorable interactions with the Gα helical domain.

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References

  1. Sprang SR (1997) G protein mechanisms: insights from structural analysis. Annu Rev Biochem 66:639–678

    Article  CAS  PubMed  Google Scholar 

  2. Oldham WM, Hamm HE (2008) Heterotrimeric G protein activation by G-protein-coupled receptors. Nat Rev Mol Cell Biol 9(1):60–71

    Article  CAS  PubMed  Google Scholar 

  3. Berman DM, Kozasa T, Gilman AG (1996) The GTPase-activating protein RGS4 stabilizes the transition state for nucleotide hydrolysis. J Biol Chem 271(44):27209–27212

    Article  CAS  PubMed  Google Scholar 

  4. Hunt TW et al (1996) RGS10 is a selective activator of Gαi GTPase activity. Nature 383(6596):175–177

    Article  CAS  PubMed  Google Scholar 

  5. Koelle MR, Horvitz HR (1996) EGL-10 regulates G protein signaling in the C. elegans nervous system and shares a conserved domain with many mammalian proteins. Cell 84(1):115–125

    Article  CAS  PubMed  Google Scholar 

  6. Siderovski DP et al (1996) A new family of regulators of G-protein-coupled receptors? Curr Biol 6(2):211–212

    Article  CAS  PubMed  Google Scholar 

  7. Watson N et al (1996) RGS family members: GTPase-activating proteins for heterotrimeric G-protein alpha-subunits. Nature 383(6596):172–175

    Article  CAS  PubMed  Google Scholar 

  8. Neubig RR, Siderovski DP (2002) Regulators of G-protein signalling as new central nervous system drug targets. Nat Rev Drug Discov 1(3):187–197

    Article  CAS  PubMed  Google Scholar 

  9. Hollinger S, Hepler JR (2002) Cellular regulation of RGS proteins: modulators and integrators of G protein signaling. Pharmacol Rev 54(3):527–559

    Article  CAS  PubMed  Google Scholar 

  10. Neitzel KL, Hepler JR (2006) Cellular mechanisms that determine selective RGS protein regulation of G protein-coupled receptor signaling. Semin Cell Dev Biol 17(3):383–389

    Article  CAS  PubMed  Google Scholar 

  11. Hurst JH, Hooks SB (2009) Regulator of G-protein signaling (RGS) proteins in cancer biology. Biochem Pharmacol 78(10):1289–1297

    Article  CAS  PubMed  Google Scholar 

  12. Kimple AJ et al (2011) Regulators of G-protein signaling and their Galpha substrates: promises and challenges in their use as drug discovery targets. Pharmacol Rev 63(3):728–749

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Ross EM, Wilkie TM (2000) GTPase-activating proteins for heterotrimeric G proteins: regulators of G protein signaling (RGS) and RGS-like proteins. Annu Rev Biochem 69:795–827

    Article  CAS  PubMed  Google Scholar 

  14. Squires KE et al (2018) Genetic analysis of rare human variants of regulators of G protein signaling proteins and their role in human physiology and disease. Pharmacol Rev 70(3):446–474

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Masuho I et al (2020) A global map of G protein signaling regulation by RGS proteins. Cell 183(2):503–521 (e19)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Glick JL et al (1998) RGSZ1, a Gz-selective regulator of G protein signaling whose action is sensitive to the phosphorylation state of Gzalpha. J Biol Chem 273(40):26008–26013

    Article  CAS  PubMed  Google Scholar 

  17. Kosloff M et al (2011) Integrating energy calculations with functional assays to decipher the specificity of G protein-RGS protein interactions. Nat Struct Mol Biol 18(7):846–853

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Salem-Mansour D et al (2018) Structural motifs in the RGS RZ subfamily combine to attenuate interactions with Gα subunits. Biochem Biophys Res Commun 503(4):2736–2741

    Article  CAS  PubMed  Google Scholar 

  19. Lan KL et al (2000) Rapid kinetics of regulator of G-protein signaling (RGS)-mediated Gαi and Gαo deactivation. Gα specificity of RGS4 AND RGS7. J Biol Chem 275(43):33497–33503

    Article  CAS  PubMed  Google Scholar 

  20. Hooks SB et al (2003) RGS6, RGS7, RGS9, and RGS11 stimulate GTPase activity of Gi family G-proteins with differential selectivity and maximal activity. J Biol Chem 278(12):10087–10093

    Article  CAS  PubMed  Google Scholar 

  21. Masuho I, Xie K, Martemyanov KA (2013) Macromolecular composition dictates receptor and G protein selectivity of regulator of G protein signaling (RGS) 7 and 9–2 protein complexes in living cells. J Biol Chem 288(35):25129–25142

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Israeli R et al (2019) RGS6 and RGS7 discriminate between the highly similar Gαi and Gαo proteins using a two-tiered specificity strategy. J Mol Biol 431(17):3302–3311

    Article  CAS  PubMed  Google Scholar 

  23. Snow BE et al (1998) GTPase activating specificity of RGS12 and binding specificity of an alternatively spliced PDZ (PSD-95/Dlg/ZO-1) domain. J Biol Chem 273(28):17749–17755

    Article  CAS  PubMed  Google Scholar 

  24. Asli A et al (2018) “Disruptor” residues in the regulator of G protein signaling (RGS) R12 subfamily attenuate the inactivation of Galpha subunits. Sci Signal 11(534):eaan3677

    Article  PubMed  CAS  Google Scholar 

  25. Heximer SP et al (1999) G protein selectivity is a determinant of RGS2 function. J Biol Chem 274(48):34253–34259

    Article  CAS  PubMed  Google Scholar 

  26. Kimple AJ et al (2009) Structural determinants of G-protein alpha subunit selectivity by regulator of G-protein signaling 2 (RGS2). J Biol Chem 284(29):19402–19411

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Kasom M et al (2018) Interplay between negative and positive design elements in Gα helical domains of G proteins determines interaction specificity toward RGS2. Biochem J 475(14):2293–2304

    Article  CAS  PubMed  Google Scholar 

  28. Natochin M, McEntaffer RL, Artemyev NO (1998) Mutational analysis of the Asn residue essential for RGS protein binding to G-proteins. J Biol Chem 273(12):6731–6735

    Article  CAS  PubMed  Google Scholar 

  29. Wieland T et al (2000) Polarity exchange at the interface of regulators of G protein signaling with G protein alpha-subunits. J Biol Chem 275(37):28500–28506

    Article  CAS  PubMed  Google Scholar 

  30. Moratz C et al (2000) Regulator of G protein signaling 1 (RGS1) markedly impairs Gαi signaling responses of B lymphocytes. J Immunol 164(4):1829–1838

    Article  CAS  PubMed  Google Scholar 

  31. Cladman W, Chidiac P (2002) Characterization and comparison of RGS2 and RGS4 as GTPase-activating proteins for m2 muscarinic receptor-stimulated G(i). Mol Pharmacol 62(3):654–659

    Article  CAS  PubMed  Google Scholar 

  32. Derrien A et al (2003) Src-mediated RGS16 tyrosine phosphorylation promotes RGS16 stability. J Biol Chem 278(18):16107–16116

    Article  CAS  PubMed  Google Scholar 

  33. Cohen SP et al (2012) Regulator of G-protein signaling-21 (RGS21) is an inhibitor of bitter gustatory signaling found in lingual and airway epithelia. J Biol Chem 287(50):41706–41719

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Tesmer JJ et al (1997) Structure of RGS4 bound to AlF4–activated Gia1: stabilization of the transition state for GTP hydrolysis. Cell 89(2):251–261

    Article  CAS  PubMed  Google Scholar 

  35. Sprang SR, Chen Z, Du X (2007) Structural basis of effector regulation and signal termination in heterotrimeric Gα proteins. Adv Protein Chem 74:1–65

    Article  CAS  PubMed  Google Scholar 

  36. Mann D et al (2016) Mechanism of the intrinsic arginine finger in heterotrimeric G proteins. Proc Natl Acad Sci USA 113(50):E8041–E8050

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Srinivasa SP et al (1998) Mechanism of RGS4, a GTPase-activating protein for G protein alpha subunits. J Biol Chem 273(3):1529–1533

    Article  CAS  PubMed  Google Scholar 

  38. Posner BA et al (1999) Modulation of the affinity and selectivity of RGS protein interaction with Gα subunits by a conserved asparagine/serine residue. Biochemistry 38(24):7773–7779

    Article  CAS  PubMed  Google Scholar 

  39. Slep KC et al (2008) Molecular architecture of Gαo and the structural basis for RGS16-mediated deactivation. Proc Natl Acad Sci USA 105(17):6243–6248

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Soundararajan M et al (2008) Structural diversity in the RGS domain and its interaction with heterotrimeric G protein alpha-subunits. Proc Natl Acad Sci USA 105(17):6457–6462

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Nance MR et al (2013) Structural and functional analysis of the regulator of G protein signaling 2-Gαq complex. Structure 21(3):438–448

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Taylor VG, Bommarito PA, Tesmer JJ (2016) Structure of the regulator of G protein signaling 8 (RGS8)-Gαq complex: molecular basis for Gα selectivity. J Biol Chem 291(10):5138–5145

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Coleman DE, Sprang SR (1998) Crystal structures of the G protein Giα1 complexed with GDP and Mg2+: a crystallographic titration experiment. Biochemistry 37(41):14376–14385

    Article  CAS  PubMed  Google Scholar 

  44. Owen VJ et al (2001) Expression of RGS3, RGS4 and Giα2 in acutely failing donor hearts and end-stage heart failure. Eur Heart J 22(12):1015–1020

    Article  CAS  PubMed  Google Scholar 

  45. Li H et al (2010) Regulator of G protein signaling 5 protects against cardiac hypertrophy and fibrosis during biomechanical stress of pressure overload. Proc Natl Acad Sci USA 107(31):13818–13823

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Ooe A, Kato K, Noguchi S (2007) Possible involvement of CCT5, RGS3, and YKT6 genes up-regulated in p53-mutated tumors in resistance to docetaxel in human breast cancers. Breast Cancer Res Treat 101(3):305–315

    Article  CAS  PubMed  Google Scholar 

  47. Ganss R (2015) Keeping the balance right: regulator of G protein signaling 5 in vascular physiology and pathology. Prog Mol Biol Transl Sci 133:93–121

    Article  PubMed  Google Scholar 

  48. Furuya M et al (2004) Expression of regulator of G protein signalling protein 5 (RGS5) in the tumour vasculature of human renal cell carcinoma. J Pathol 203(1):551–558

    Article  CAS  PubMed  Google Scholar 

  49. Altman MK et al (2012) Regulator of G-protein signaling 5 reduces HeyA8 ovarian cancer cell proliferation and extends survival in a murine tumor model. Biochem Res Int 2012:518437

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  50. Hu M et al (2013) Over-expression of regulator of G protein signaling 5 promotes tumor metastasis by inducing epithelial-mesenchymal transition in hepatocellular carcinoma cells. J Surg Oncol 108(3):192–196

    Article  CAS  PubMed  Google Scholar 

  51. Druey KM (2009) Regulation of G-protein-coupled signaling pathways in allergic inflammation. Immunol Res 43(1–3):62–76

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Hwang IY et al (2013) Rgs13 constrains early B cell responses and limits germinal center sizes. PLoS ONE 8(3):e60139

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Wang JH et al (2013) Extension of the germinal center stage of B cell development promotes autoantibodies in BXD2 mice. Arthritis Rheum 65(10):2703–2712

    CAS  PubMed  PubMed Central  Google Scholar 

  54. Williams JW et al (2013) RGS3 controls T lymphocyte migration in a model of Th2-mediated airway inflammation. Am J Physiol Lung Cell Mol Physiol 305(10):L693-701

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Xie Z, Chan EC, Druey KM (2016) R4 regulator of G protein signaling (RGS) proteins in inflammation and immunity. AAPS J 18(2):294–304

    Article  CAS  PubMed  Google Scholar 

  56. O’Brien JB, Wilkinson JC, Roman DL (2019) Regulator of G-protein signaling (RGS) proteins as drug targets: progress and future potentials. J Biol Chem 294(49):18571–18585

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Senese NB et al (2020) Regulator of G-protein signaling (RGS) protein modulation of opioid receptor signaling as a potential target for pain management. Front Mol Neurosci 13:5

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. DiGiacomo V et al (2020) Probing the mutational landscape of regulators of G protein signaling proteins in cancer. Sci Signal 13(617):eaax8620

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Bakhman A et al (2019) Residue-level determinants of angiopoietin-2 interactions with its receptor Tie2. Proteins 87(3):185–197

    Article  CAS  PubMed  Google Scholar 

  60. Shushan A, Kosloff M (2021) Structural design principles for specific ultra-high affinity interactions between colicins/pyocins and immunity proteins. Sci Rep 11(1):3789

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Gibson DG et al (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 6(5):343–345

    Article  CAS  PubMed  Google Scholar 

  62. Sun D et al (2013) AAscan, PCRdesign and MutantChecker: a suite of programs for primer design and sequence analysis for high-throughput scanning mutagenesis. PLoS ONE 8(10):e78878

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Wang J et al (1997) A GTPase-activating protein for the G protein Gαz: identification, purification, and mechanism of action. J Biol Chem 272(9):5732–5740

    Article  CAS  PubMed  Google Scholar 

  64. Ross EM (2002) Quantitative assays for GTPase-activating proteins. Methods Enzymol 344:601–617

    Article  CAS  PubMed  Google Scholar 

  65. Mukhopadhyay S, Ross EM (1999) Rapid GTP binding and hydrolysis by G(q) promoted by receptor and GTPase-activating proteins. Proc Natl Acad Sci USA 96(17):9539–9544

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank John Kehrl (NIH) for RGS3, RGS5, and RGS13 clones, Vadim Arshavsky for the Gαo clone, and Anna Bakhman for technical assistance.

Funding

This work was supported by grants from the Israel Science Foundation (Grant Numbers: 1454/13, 1959/13, 2155/15), the Canadian Institutes of Health Research (CIHR), the International Development Research Centre (IDRC), the Israel Science Foundation (ISF), and the Azrieli Foundation (Grant Number 3512/19) and the DS Research Center at the University of Haifa. The authors acknowledge the contributions of COST Actions CM-1207 (GLISTEN), CA-15126 (ARBIEU), and CA-18133 (ERNEST) and iNEXT actions PID 4101 and 6623 to this work.

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  1. Ali Asli and Sabreen Higazy-Mreih contributed equally to this work.

Authors and Affiliations

  1. The Department of Human Biology, Faculty of Natural Science, University of Haifa, 199 Aba Khoushy Ave., Mt. Carmel, 3498838, Haifa, Israel

    Ali Asli, Sabreen Higazy-Mreih, Meirav Avital-Shacham & Mickey Kosloff

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  1. Ali Asli
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  2. Sabreen Higazy-Mreih
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  3. Meirav Avital-Shacham
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  4. Mickey Kosloff
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Contributions

AA designed the research, conducted most of the experiments and structural analysis, analyzed results, and wrote the paper. SHM conducted experiments, analyzed results, and contributed to the paper. MAS conducted experiments, supervised lab work, analyzed results, and contributed to the paper. MK designed and supervised the research and analysis and wrote the paper. All authors were involved in the writing of the paper and approved the final version.

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Correspondence to Mickey Kosloff.

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Asli, A., Higazy-Mreih, S., Avital-Shacham, M. et al. Residue-level determinants of RGS R4 subfamily GAP activity and specificity towards the Gi subfamily. Cell. Mol. Life Sci. 78, 6305–6318 (2021). https://doi.org/10.1007/s00018-021-03898-4

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