Agonist-Induced Formation of Unproductive Receptor-G12 Complexes Najeah Okashaha, Shane C

Agonist-induced formation of unproductive receptor-G12 complexes Najeah Okashaha, Shane C. Wrightb, Kouki Kawakamic, Signe Mathiasend,e,f, Joris Zhoub, Sumin Lua, Jonathan A. Javitchd,e,f, Asuka Inouec, Michel Bouvierb, and Nevin A. Lamberta,1 aDepartment of Pharmacology and Toxicology, Medical College of Georgia, Augusta University, Augusta, GA 30912; bInstitute for Research in Immunology and Cancer, Department of Biochemistry and Molecular Medicine, Université de Montréal, Montréal, QC H3T 1J4, Canada; cGraduate School of Pharmaceutical Sciences, Tohoku University, 980-8578 Sendai, Japan; dDepartment of Psychiatry, Columbia University Vagelos College of Physicians and Surgeons, New York, NY 10032; eDepartment of Pharmacology, Columbia University Vagelos College of Physicians and Surgeons, New York, NY 10032; and fDivision of Molecular Therapeutics, New York State Psychiatric Institute, New York, NY 10032 Edited by Brian K. Kobilka, Stanford University School of Medicine, Stanford, CA, and approved July 24, 2020 (received for review February 28, 2020) G proteins are activated when they associate with G protein- association does not always promote G protein activation and coupled receptors (GPCRs), often in response to agonist-mediated may in some circumstances inhibit downstream signaling. receptor activation. It is generally thought that agonist-induced receptor-G protein association necessarily promotes G protein acti- Results vation and, conversely, that activated GPCRs do not interact with V2R Interacts with G12 Heterotrimers. Conventional GPCR-G pro- G proteins that they do not activate. Here we show that GPCRs can tein coupling is understood as an allosteric interaction where an form agonist-dependent complexes with G proteins that they do agonist-bound active receptor mediates GDP release by stabi- not activate. Using cell-based bioluminescence resonance energy lizing the nucleotide-free state of an associated Gα subunit (3–5). transfer (BRET) and luminescence assays we find that vasopressin Receptor complexes with nucleotide-free G proteins are quite V2 receptors (V2R) associate with both Gs and G12 heterotrimers transient when guanine nucleotides are present at concentrations when stimulated with the agonist arginine vasopressin (AVP). How- similar to those found in cells, but are stabilized when guanine ever, unlike V2R-Gs complexes, V2R-G12 complexes are not destabi- nucleotides are absent. In order to observe allosteric coupling we lized by guanine nucleotides and do not promote G12 activation. monitored receptor-G protein association under conditions that Activating V2R does not lead to signaling responses downstream allowed us to control both ligand binding to the receptor and PHARMACOLOGY of G12 activation, but instead inhibits basal G12-mediated signaling, nucleotide binding to the G protein. We used bioluminescence presumably by sequestering G12 heterotrimers. Overexpressing G12 resonance energy transfer (BRET) between GPCRs fused to inhibits G protein receptor kinase (GRK) and arrestin recruitment to Renilla luciferase (Rluc8) and Gβ1 and Gγ2 subunits fused to V2R and receptor internalization. Formyl peptide (FPR1 and FPR2) complementary fragments of Venus fluorescent protein (6–8) to and Smoothened (Smo) receptors also form complexes with G12 that monitor receptor-G protein association. These components and are insensitive to nucleotides, suggesting that unproductive GPCR- unlabeled Gα subunits were transfected into HEK 293 cells in G12 complexes are not unique to V2R. These results indicate that which most of the endogenous G proteins had been deleted agonist-dependent receptor-G protein association does not always using CRISPR/Cas9-mediated gene editing (9, 10). In order to lead to G protein activation and may in fact inhibit G protein control nucleotide binding to G proteins, cells were per- activation. meabilized and either supplemented with nucleotides or treated with apyrase to remove residual nucleotides. GPCR | ternary complex | G protein-coupled receptor | arrestin Significance protein-coupled receptors (GPCRs) mediate important physiological activities and exert most of their effects G G protein-coupled receptors (GPCRs) are targeted by a large through activation of G proteins. In the conventional model of fraction of approved drugs and regulate many important cel- coupling, unliganded receptors are poor recruiters and activators lular processes. Association of GPCRs with heterotrimeric G of G proteins, whereas agonist-bound GPCRs take on more proteins in response to agonist activation is thought to in- active conformations that effectively recruit G protein hetero- variably lead to G protein activation. We find instead that G trimers (1, 2). Productive receptor-G protein association pro- 12 heterotrimers can associate with agonist-bound receptors in a motes GDP release by stabilizing the nucleotide-free state of the manner that does not lead to activation. These unproductive Gα subunit, which in turn allows GTP binding, G protein acti- agonist–receptor-G protein ternary complexes sequester G vation, and downstream signaling (3, 4). According to this model, 12 heterotrimers and thus inhibit rather than support G signal- agonist-dependent GPCR-G protein complex formation is es- 12 ing. These findings reveal a mechanism whereby agonist acti- sentially synonymous with G protein activation. Four families of vation of GPCRs can inhibit as well as promote G protein G proteins (G ,G ,G , and G ) can be activated, and each s i/o q/11 12/13 signaling. leads to a distinct set of downstream signaling outcomes. It is generally thought that selection of G protein subtypes by GPCRs Author contributions: N.O., S.C.W., A.I., M.B., and N.A.L. designed research; N.O., S.C.W., occurs at the receptor-G protein association step, such that re- K.K., S.M., J.Z., and S.L. performed research; A.I. contributed new reagents/analytic tools; ceptors interact with and activate cognate G protein subtypes N.O., S.C.W., A.I., and N.A.L. analyzed data; and N.O., J.A.J., A.I., M.B., and N.A.L. wrote and do not interact with noncognate G protein subtypes. Here the paper. we find that agonist-dependent GPCR-G protein association can The authors declare no competing interest. occur without promoting subsequent G protein activation, thus, This article is a PNAS Direct Submission. whether a G protein subtype is activated can be determined after This open access article is distributed under Creative Commons Attribution-NonCommercial- initial receptor-G protein engagement. Moreover, noncognate G NoDerivatives License 4.0 (CC BY-NC-ND). proteins can impede downstream events, perhaps by competing 1To whom correspondence may be addressed. Email: [email protected]. with other intracellular transducers for access to activated re- This article contains supporting information online at https://www.pnas.org/lookup/suppl/ ceptors. These findings revise the standard model of G protein doi:10.1073/pnas.2003787117/-/DCSupplemental. coupling by indicating that agonist-induced GPCR-G protein www.pnas.org/cgi/doi/10.1073/pnas.2003787117 PNAS Latest Articles | 1of8 Downloaded by guest on September 26, 2021 Because receptor-G protein complexes are transient it can be observed surprisingly robust agonist-induced BRET between V2R difficult to detect agonist-induced BRET signals between re- and G12 heterotrimers in the presence of GDP that was not en- ceptors and G proteins when guanine nucleotides are present hanced by nucleotide depletion (Fig. 1A). These results suggest (6). For example, arginine vasopressin (AVP) did not detectably that AVP-bound V2R can form complexes with GDP-bound G12 increase BRET between vasopressin V2 receptors (V2R) and Gs heterotrimers that do not progress to the nucleotide-free state and heterotrimers in the presence of GDP (Fig. 1A). In contrast, AVP therefore are not stabilized when GDP is removed (Fig. 1B). As an produced large BRET increases in the absence of nucleotides index of allosteric coupling we divide the increase in BRET pro- Δ (Fig. 1A). Stabilization of agonist–receptor-G protein complexes duced by agonist in the presence of GDP ( BRETag) by the in- α crease in BRET produced by the combined effect of agonist and when G subunits are nucleotide-free indicates conventional Δ “productive” allosteric coupling and predicts that agonist-bound nucleotide depletion ( BRETag+apy), and refer to this index as receptors will promote GDP release and G protein activation the GDP-resistance ratio, or RGDP.RGDP values that are less than 1 indicate conventional productive coupling, whereas an R under physiological conditions. Nucleotide-free conditions also GDP value of 1 indicates nucleotide-resistant or unproductive coupling. enhanced AVP-induced BRET between V RandG or G het- 2 i1 q R for V R and G was 0.08 ± 0.12 (mean ± SD; n = 6), erotrimers (SI Appendix,Fig.S1). These results are consistent with GDP 2 s whereas RGDP for V2R and G12 was 1.01 ± 0.06 (n = 6). In cognate V2R activation of Gs and Gq heterotrimers (11, 12) and contrast to V2R, we observed more conventional productive predict some ability to activate Gi1 heterotrimers. In contrast, we coupling of both endothelin A (ETA)andthromboxaneA2 (TP) receptors with G12 heterotrimers, with RGDP values of 0.66 ± 0.09 (n = 3) and 0.55 ± 0.09 (n = 6), respectively. Both of these re- V2R A ceptors also coupled productively with Gq heterotrimers, with 0.2 0.2 ± n = ± n = Gs G12 RGDP values of 0.29 0.08 ( 3) and 0.11 0.07 ( 6), re- ns spectively (SI Appendix,Fig.S2). Receptors that couple to one member of a Gα subunit family can usually couple to other members of the same family. There- fore, we examined V2R coupling to G13 heterotrimers, the other net BRET member of the G12/13 family (13). We found that stimulation with AVP increased BRET between V2RandG13 heterotrimers in the 0.0 0.0 presence of GDP (SI Appendix,Fig.S3). However, unlike what we - AVP - AVP - AVP - AVP observed with G12, these responses were enhanced by nucleotide GDP apy GDP apy depletion (RGDP = 0.67 ± 0.08; n = 4), indicating productive B coupling, consistent with weak V2R-mediated activation of G13 AVP (14).

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