Multiple potassium channel tetramerization domain (KCTD) family members interact with Gβγ, with effects on cAMP signaling
2023; Elsevier BV; Volume: 299; Issue: 3 Linguagem: Inglês
10.1016/j.jbc.2023.102924
ISSN1083-351X
AutoresDouglas C. Sloan, Casey E. Cryan, Brian S. Muntean,
Tópico(s)Protein Kinase Regulation and GTPase Signaling
ResumoG protein–coupled receptors (GPCRs) initiate an array of intracellular signaling programs by activating heterotrimeric G proteins (Gα and Gβγ subunits). Therefore, G protein modifiers are well positioned to shape GPCR pharmacology. A few members of the potassium channel tetramerization domain (KCTD) protein family have been found to adjust G protein signaling through interaction with Gβγ. However, comprehensive details on the KCTD interaction with Gβγ remain unresolved. Here, we report that nearly all the 25 KCTD proteins interact with Gβγ. In this study, we screened Gβγ interaction capacity across the entire KCTD family using two parallel approaches. In a live cell bioluminescence resonance energy transfer–based assay, we find that roughly half of KCTD proteins interact with Gβγ in an agonist-induced fashion, whereas all KCTD proteins except two were found to interact through coimmunoprecipitation. We observed that the interaction was dependent on an amino acid hot spot in the C terminus of KCTD2, KCTD5, and KCTD17. While KCTD2 and KCTD5 require both the Bric-à-brac, Tramtrack, Broad complex domain and C-terminal regions for Gβγ interaction, we uncovered that the KCTD17 C terminus is sufficient for Gβγ interaction. Finally, we demonstrated the functional consequence of the KCTD–Gβγ interaction by examining sensitization of the adenylyl cyclase–cAMP pathway in live cells. We found that Gβγ-mediated sensitization of adenylyl cyclase 5 was blunted by KCTD. We conclude that the KCTD family broadly engages Gβγ to shape GPCR signal transmission. G protein–coupled receptors (GPCRs) initiate an array of intracellular signaling programs by activating heterotrimeric G proteins (Gα and Gβγ subunits). Therefore, G protein modifiers are well positioned to shape GPCR pharmacology. A few members of the potassium channel tetramerization domain (KCTD) protein family have been found to adjust G protein signaling through interaction with Gβγ. However, comprehensive details on the KCTD interaction with Gβγ remain unresolved. Here, we report that nearly all the 25 KCTD proteins interact with Gβγ. In this study, we screened Gβγ interaction capacity across the entire KCTD family using two parallel approaches. In a live cell bioluminescence resonance energy transfer–based assay, we find that roughly half of KCTD proteins interact with Gβγ in an agonist-induced fashion, whereas all KCTD proteins except two were found to interact through coimmunoprecipitation. We observed that the interaction was dependent on an amino acid hot spot in the C terminus of KCTD2, KCTD5, and KCTD17. While KCTD2 and KCTD5 require both the Bric-à-brac, Tramtrack, Broad complex domain and C-terminal regions for Gβγ interaction, we uncovered that the KCTD17 C terminus is sufficient for Gβγ interaction. Finally, we demonstrated the functional consequence of the KCTD–Gβγ interaction by examining sensitization of the adenylyl cyclase–cAMP pathway in live cells. We found that Gβγ-mediated sensitization of adenylyl cyclase 5 was blunted by KCTD. We conclude that the KCTD family broadly engages Gβγ to shape GPCR signal transmission. G protein–coupled receptors (GPCRs) represent one of the most prominent mechanisms for cellular communication, controlling key physiological processes in almost every mammalian cell and tissue type (1Wettschureck N. Offermanns S. Mammalian G proteins and their cell type specific functions.Physiol. Rev. 2005; 85: 1159-1204Crossref PubMed Scopus (870) Google Scholar). In a prototypic series of events, ligand-activated GPCRs induce the mobilization of heterotrimeric G proteins (Gα and obligatory Gβγ dimers) to engage effector molecules triggering downstream events (2Lambert N.A. Dissociation of heterotrimeric g proteins in cells.Sci. Signal. 2008; 1: re5Crossref PubMed Scopus (69) Google Scholar, 3Pierce K.L. Premont R.T. Lefkowitz R.J. Seven-transmembrane receptors.Nat. Rev. Mol. Cell Biol. 2002; 3: 639-650Crossref PubMed Scopus (2163) Google Scholar). Thus, modulation of active G protein lifetime critically dictates signaling magnitude and duration (4Anderson G.R. Posokhova E. Martemyanov K.A. The R7 RGS protein family: multi-subunit regulators of neuronal G protein signaling.Cell Biochem. Biophys. 2009; 54: 33-46Crossref PubMed Scopus (114) Google Scholar). In particular, Gβγ engages a host of effectors (5Dupre D.J. Robitaille M. Rebois R.V. Hebert T.E. 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This is exemplified in the case of Gβγ gating of K+ flux through G protein–gated inwardly rectifying potassium (GIRK) channels (9Luscher C. Slesinger P.A. Emerging roles for G protein-gated inwardly rectifying potassium (GIRK) channels in health and disease.Nat. Rev. Neurosci. 2010; 11: 301-315Crossref PubMed Scopus (454) Google Scholar). The GPCR–GIRK signaling axis is finely tuned by proteins that either promote or reduce Gβγ availability (10Luo H. Marron Fernandez de Velasco E. Wickman K. Neuronal G protein-gated K(+) channels.Am. J. Physiol. Cell Physiol. 2022; 323: C439-C460Crossref PubMed Scopus (11) Google Scholar), with one example pertaining to the binding of Gβγ by certain potassium channel tetramerization domain (KCTD) proteins (11Turecek R. Schwenk J. Fritzius T. Ivankova K. Zolles G. Adelfinger L. et al.Auxiliary GABAB receptor subunits uncouple G protein betagamma subunits from effector channels to induce desensitization.Neuron. 2014; 82: 1032-1044Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 12Zheng S. Abreu N. Levitz J. Kruse A.C. Structural basis for KCTD-mediated rapid desensitization of GABAB signalling.Nature. 2019; 567: 127-131Crossref PubMed Scopus (48) Google Scholar). The KCTD family consists of 25 proteins that contain great diversity outside a structurally similar Bric-à-brac, Tramtrack, Broad complex (BTB) domain, which organizes KCTD complex oligomerization (13Schwenk J. Metz M. Zolles G. Turecek R. Fritzius T. Bildl W. et al.Native GABA(B) receptors are heteromultimers with a family of auxiliary subunits.Nature. 2010; 465: 231-235Crossref PubMed Scopus (248) Google Scholar, 14Teng X. Aouacheria A. Lionnard L. Metz K.A. Soane L. Kamiya A. et al.Kctd: a new gene family involved in neurodevelopmental and neuropsychiatric disorders.CNS Neurosci. Ther. 2019; 25: 887-902Crossref PubMed Scopus (55) Google Scholar). The function of most KCTD proteins have remained relatively obscure, despite numerous ties to pathophysiological conditions (14Teng X. Aouacheria A. Lionnard L. Metz K.A. Soane L. Kamiya A. et al.Kctd: a new gene family involved in neurodevelopmental and neuropsychiatric disorders.CNS Neurosci. Ther. 2019; 25: 887-902Crossref PubMed Scopus (55) Google Scholar, 15Angrisani A. Di Fiore A. De Smaele E. Moretti M. The emerging role of the KCTD proteins in cancer.Cell Commun. Signal. 2021; 19: 56Crossref PubMed Scopus (24) Google Scholar). In addition to regulating the GABAB–Gβγ–GIRK signaling axis by KCTD12 and KCTD16 (11Turecek R. Schwenk J. Fritzius T. Ivankova K. Zolles G. Adelfinger L. et al.Auxiliary GABAB receptor subunits uncouple G protein betagamma subunits from effector channels to induce desensitization.Neuron. 2014; 82: 1032-1044Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 12Zheng S. Abreu N. Levitz J. Kruse A.C. Structural basis for KCTD-mediated rapid desensitization of GABAB signalling.Nature. 2019; 567: 127-131Crossref PubMed Scopus (48) Google Scholar), there is a growing appreciation that numerous KCTDs serve as adapters that scaffold Cullin3 to mediate ubiquitination of target proteins (16Azizieh R. Orduz D. Van Bogaert P. Bouschet T. Rodriguez W. Schiffmann S.N. et al.Progressive myoclonic epilepsy-associated gene KCTD7 is a regulator of potassium conductance in neurons.Mol. Neurobiol. 2011; 44: 111-121Crossref PubMed Scopus (53) Google Scholar, 17Bayon Y. Trinidad A.G. de la Puerta M.L. Del Carmen Rodriguez M. Bogetz J. 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Kawakami Y. Kiyono T. Yonemura S. Kawamura Y. Era S. et al.Ubiquitin-proteasome system controls ciliogenesis at the initial step of axoneme extension.Nat. Commun. 2014; 5: 5081Crossref PubMed Scopus (106) Google Scholar, 21Cho H.J. Ryu K.J. Baek K.E. Lim J. Kim T. Song C.Y. et al.Cullin 3/KCTD5 promotes the Ubiqutination of rho guanine nucleotide dissociation inhibitor 1 and regulates its stability.J. Microbiol. Biotechnol. 2020; 30: 1488-1494Crossref PubMed Google Scholar, 22He H. Peng Y. Fan S. Chen Y. Zheng X. Li C. Cullin3/KCTD5 induces monoubiquitination of DeltaNp63alpha and impairs its activity.FEBS Lett. 2018; 592: 2334-2340Crossref PubMed Scopus (8) Google Scholar). Curiously, the best characterized substrate for ubiquitination is Gβγ (23Brockmann M. Blomen V.A. Nieuwenhuis J. Stickel E. Raaben M. Bleijerveld O.B. et al.Genetic wiring maps of single-cell protein states reveal an off-switch for GPCR signalling.Nature. 2017; 546: 307-311Crossref PubMed Scopus (77) Google Scholar), which is mediated through interaction with KCTD2 and KCTD5 (24Young B.D. Sha J. Vashisht A.A. Wohlschlegel J.A. Human multisubunit E3 ubiquitin ligase required for heterotrimeric G-protein beta-subunit ubiquitination and downstream signaling.J. Proteome Res. 2021; 20: 4318-4330Crossref PubMed Scopus (4) Google Scholar). Indeed, loss of KCTD2 and KCTD5 (as well as KCTD17) leads to enhanced Gβγ-dependent second messenger signaling downstream through the cAMP pathway (25Muntean B.S. Marwari S. Li X. Sloan D.C. Young B.D. Wohlschlegel J.A. et al.Members of the KCTD family are major regulators of cAMP signaling.Proc. Natl. Acad. Sci. U. S. A. 2022; 119e2119237119Crossref PubMed Scopus (8) Google Scholar). Despite substantial functional information resulting from interactions between Gβγ with a few KCTDs (12Zheng S. Abreu N. Levitz J. Kruse A.C. Structural basis for KCTD-mediated rapid desensitization of GABAB signalling.Nature. 2019; 567: 127-131Crossref PubMed Scopus (48) Google Scholar), the molecular determinants underpinning such interaction are still unclear. Moreover, investigation into the remaining KCTD family toward Gβγ has yet to be defined. In this study, we utilized two independent approaches to screen Gβγ interaction profiles across the entire KCTD family. Despite considerable diversity between KCTDs, we report that nearly every KCTD interacts with Gβγ though immunoprecipitation (IP), and about half of the KCTD family interact in an agonist-induced fashion in a bioluminescence resonance energy transfer (BRET)–based assay. We then utilized a subset of KCTD to further investigate rules of engagement for selectivity for interaction with Gβγ. We demonstrate how these principles enable KCTD2/5/9/17 to modulate Gβγ-dependent signal transmission in live cells. Finally, we report that various KCTDs modulate efficacy of cAMP signaling in primary striatal neurons. Combined functional and structural data strongly support KCTD engagement with Gβγ following GPCR activation (11Turecek R. Schwenk J. Fritzius T. Ivankova K. Zolles G. Adelfinger L. et al.Auxiliary GABAB receptor subunits uncouple G protein betagamma subunits from effector channels to induce desensitization.Neuron. 2014; 82: 1032-1044Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 12Zheng S. Abreu N. Levitz J. Kruse A.C. Structural basis for KCTD-mediated rapid desensitization of GABAB signalling.Nature. 2019; 567: 127-131Crossref PubMed Scopus (48) Google Scholar, 23Brockmann M. Blomen V.A. Nieuwenhuis J. Stickel E. Raaben M. Bleijerveld O.B. et al.Genetic wiring maps of single-cell protein states reveal an off-switch for GPCR signalling.Nature. 2017; 546: 307-311Crossref PubMed Scopus (77) Google Scholar, 26Zuo H. Glaaser I. Zhao Y. Kurinov I. Mosyak L. Wang H. et al.Structural basis for auxiliary subunit KCTD16 regulation of the GABAB receptor.Proc. Natl. Acad. Sci. U. S. A. 2019; 116: 8370-8379Crossref PubMed Scopus (28) Google Scholar). Therefore, we began with an unbiased functional evaluation to determine which KCTD proteins could interact with Gβγ dimers. We devised a BRET assay to monitor GPCR agonist–induced interaction of KCTD with Gβγ in live cells. For the BRET donor, we fused Nanoluciferase (Nluc) to the C terminus of each full-length KCTD. Upon transfection in human embryonic kidney 293 (HEK293) cells and exposure to Nano-Glo substrate, each Nluc construct yielded similar bioluminescence intensity suggesting relatively equal KCTD expression level (Fig. 1A). We then utilized the well-characterized bimolecular fluorescence complementation Venus fluorophore split between Gβ1 (Venus 156-239-Gβ1) and Gγ2 (Venus 1-155-Gγ2) as the BRET acceptor strategy (27Hollins B. Kuravi S. Digby G.J. Lambert N.A. The c-terminus of GRK3 indicates rapid dissociation of G protein heterotrimers.Cell Signal. 2009; 21: 1015-1021Crossref PubMed Scopus (105) Google Scholar). In our assay, HEK293 cells were transiently transfected with KCTD-Nluc, Gβγ–Venus, D2 dopamine receptor (D2R), and GαoA (Fig. 1B). GPCR signal transmission was initiated by D2R activation with dopamine. We recorded the agonist-induced BRET response after 5 min and compared with basal readings (Fig. 1C). As a reference, we performed control experiments with Nluc fused to the C terminus of the GRK3 effector (GRK3ct), which exhibits nanomolar affinity for Gβγ (28Pitcher J.A. Inglese J. Higgins J.B. Arriza J.L. Casey P.J. Kim C. et al.Role of beta gamma subunits of G proteins in targeting the beta-adrenergic receptor kinase to membrane-bound receptors.Science. 1992; 257: 1264-1267Crossref PubMed Scopus (640) Google Scholar) and readily reports agonist-induced association with Gβγ–Venus (29Masuho I. Ostrovskaya O. Kramer G.M. Jones C.D. Xie K. Martemyanov K.A. Distinct profiles of functional discrimination among G proteins determine the actions of G protein-coupled receptors.Sci. Signal. 2015; 8: ra123Crossref PubMed Scopus (158) Google Scholar, 30Muntean B.S. Martemyanov K.A. Association with the plasma membrane is sufficient for potentiating catalytic activity of regulators of G protein signaling (RGS) proteins of the R7 subfamily.J. Biol. Chem. 2016; 291: 7195-7204Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar). Approximately half of the KCTD family exhibited an increased BRET signal after dopamine application, suggesting interaction with Gβγ (Fig. 1D). Among these were several KCTDs previously identified in complex with Gβγ (KCTD2, KCTD5, and KCTD12). On the other hand, KCTD16, which has been demonstrated to bind Gβγ (11Turecek R. Schwenk J. Fritzius T. Ivankova K. Zolles G. Adelfinger L. et al.Auxiliary GABAB receptor subunits uncouple G protein betagamma subunits from effector channels to induce desensitization.Neuron. 2014; 82: 1032-1044Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar), generated only a mild BRET response in our experiments. However, KCTD16 elicited a relatively higher basal BRET, which could indicate interaction with Gβγ prior to receptor stimulation. In addition, we revealed numerous KCTDs that had not previously been known to engage Gβγ. To gain insight toward binding patterns, we generated a phylogenetic tree of the human KCTD family aligned with a heatmap of the BRET fold change (Fig. 1E). The analysis revealed consistency in Gβγ interaction between KCTD subgroups. One exception to this observation was the lack of BRET response with KCTD9, differing from the robust signal exhibited by similar group members (KCTD2, KCTD5, and KCTD17). Given that KCTD16 yielded a small netBRET and relatively higher basal BRET, we wanted to ensure that our approach did not limit detection of KCTD–Gβγ interactions. Therefore, we next utilized IP to examine the capacity of KCTD to interact with Gβγ. In this experiment, we fused a myc tag to the carboxy terminus of the KCTD ORF for transfection into HEK293 cells in tandem with GαoA and Gβγ–Venus. Cells were then lysed followed by pulldown with a GFP antibody. We first tested whether promoting heterotrimeric G protein dissociation would enhance detection of in the KCTD–Gβγ interactions and therefore performed lysis/IP in the presence or the absence of AlF4- (Fig. 2A). For this purpose, cells were cotransfected with KCTD2 (highest netBRET) and KCTD20 (no netBRET). Probing the total lysate with an anti-myc antibody revealed similar KCTD expression level corresponding to the estimated molecular weight (Fig. 2B). Treatment with AlF4- increased the KCTD2 band intensity in the IP samples; however, KCTD20 was not detected in the IP regardless the treatment. The result fortifies the BRET observation for KCTD2/KCTD20, suggests AlF4- may enhance the detection window for KCTD–Gβγ interactions through co-IP, and demonstrates AlF4- treatment will not induce detection of false positives (at least in the case of KCTD20). Therefore, we applied the IP strategy with AlF4- to the entire KCTD family. Probing total lysates with a myc-tag antibody demonstrated expression of each KCTD near the predicted molecular weight (Fig. 2C). Stunningly, the IP samples revealed interaction of Gβγ with all but two KCTD proteins (KCTD9 and KCTD20). Although not quantitative, the IP results trend toward categories of stronger (KCTD2, 3, 5, 7, 8, 10, 15, 16, 17, 18, BTBD10, SHKBP1) and weaker (KCTD1, 4, 6, 11, 12, 19, KCNRG, TNFAIP1) interactions with Gβγ. Therefore, we examined the possibility of nonspecific interactions by repeating the Gβγ–Venus IP in parallel with Venus-transfected cells. For this experiment, we utilized three weak binders (KCTD4, KCNRG, and TNFAIP1), two strong binders (BTBD10 and KCTD18), and one nonbinder (KCTD20). While protein levels were similar in total lysates across the board, no KCTDs were detected in Venus IP, whereas all but KCTD20 were detected in the Gβγ–Venus IP (Fig. 2D). The results collectively show that while at least half of the KCTD family were observed to engage Gβγ in an agonist-induced fashion in live cells, nearly all KCTDs have the capacity for Gβγ interaction through IP. While KCTD interactions with Gβγ may vary in strength, control experiments suggest they do not appear to be false positives. We next sought to understand molecular determinants that enable broad KCTD interaction with Gβγ. Of the noninteractors, KCTD20 shares isolated similarity with BTBD10, whereas KCTD9 belongs to a subfamily that includes several members (KCTD2, KCTD5, and KCTD17) as well as conservation between other clades (Figs. 1C and S1). Therefore, we reasoned that differences in amino acids between KCTD9 and its subfamily could reveal distinct signatures that enable interaction with Gβγ. Given the paucity of information on KCTD9 interaction profiles, we started by reducing the KCTD architecture to its simplest shared domains: (i) varying length N terminus, (ii) BTB domain, (iii) remaining C terminus (Fig. 3A). Comparison of BTB domains revealed ∼50% identity between KCTD9 with KCTD2, KCTD5, or KCTD17, whereas KCTD2, KCTD5, and KCTD17 exhibited greater than 75% identity between each other (Fig. 3B). Similarly, KCTD2, KCTD5, and KCTD17 exhibit high identity between their C termini (>60%), whereas KCTD9 exhibits low C-terminal identity (∼20%) with KCTD2, KCTD5, and KCTD17 (Fig. 3B). Therefore, we hypothesized that selectivity for Gβγ interaction may be conferred by the C terminus. We made chimeras of myc-tagged KCTD9 by replacing its C terminus with that of KCTD2, KCTD5, or KCTD17. We then repeated IP experiments with Gβγ–Venus. Indeed, we observed that each KCTD9 chimera was able to interact with Gβγ (Fig. 3C). We next tested if the C terminus of KCTD9 would impede interaction with Gβγ. Thus, we made chimeras of myc-tagged KCTD2, KCTD5, and KCTD17 by replacing their C terminus with that of KCTD9. Interestingly, we found that these chimeras retained the ability to interact with Gβγ in our IP experiment (Fig. 3D), suggesting interplay between elements within the BTB and C-terminal regions of KCTD2, KCTD5, and KCTD17. We next performed a bioinformatics analysis to understand which components enable Gβγ interaction. We identified a region of charged and polar amino acids in the C terminus of KCTD2, KCTD5, and KCTD17 that were not conserved in KCTD9 (Fig. 3E). We made a myc-tagged KCTD5 mutant where we swapped these residues with the ones from KCTD9. Likewise, we replaced these residues in myc-tagged KCTD9 with their counterpart from KCTD5 and then performed the IP experiments. We found that while the KCTD5 mutant was still able to interact with Gβγ (although seemingly lesser than wildtype), the presence of the charged/polar residues on KCTD9 began to enable binding capacity for Gβγ as well (Fig. 3F). These experiments collectively suggest that the KCTD2/5/9/17 clade require key residues within both the BTB domain and C terminus in order to interact with Gβγ. We next determined if tag placement on KCTD influenced interaction with Gβγ. For this purpose, we fused mCherry to either the N terminus or C terminus of KCTD2, KCTD5, and KCTD17 (Fig. 4A). Placement of mCherry at either position yielded equivalent expression in the total lysate as well as interaction profile with Gβγ through IP (Fig. 4B). Next, we asked if the BTB domain or C terminus alone was sufficient for interaction with Gβγ–Venus in our IP experiments. We mapped these regions by generating N-terminal mCherry tags on KCTD2, KCTD5, and KCTD17 (Fig. 5A). We utilized the full-length construct as a control (mCh-FL), deleted the N terminus (mCh-ΔN), expressed only the BTB domain (mCh-BTB), or expressed only the C terminus (mCh-C-term). Starting with KCTD2, we found that the N terminus was dispensable for interaction with Gβγ (Fig. 5B). However, the BTB domain and C terminus were not sufficient for Gβγ interaction. Interestingly, coexpression of BTB domain and C terminus from separate vectors was also unable to provide interaction with Gβγ. We found that KCTD5 elicited the same interaction profile (Fig. 5C), likely owing to high degree of similarity with KCTD2. We next mapped KCTD17, which showed that while the N terminus was not required for interaction with Gβγ, the C terminus was sufficient (Fig. 5D). Intriguingly, the C terminus of KCTD17 contains a coiled-coil (CC) domain that is not conserved with other KCTDs (Fig. 5E). Therefore, we generated N-terminal mCherry fusion constructs mapping regions of the KCTD17 C terminus to investigate involvement of this region (Fig. 5F). Unfortunately, the mCherry-CC domain did not express well following transfection compared with other regions of the KCTD17 C terminus (Fig. 5G). Moreover, only the full-length KCTD17 C terminus (95–314) was able to interact with Gβγ (Fig. 5G). Thus, these interaction studies demonstrate that the KCTD N terminus is not required for interaction with Gβγ; however, the C terminus of KCTD17 is sufficient for binding in the absence of a BTB domain.Figure 5KCTD17 C terminus is sufficient for Gβγ interaction. A, scheme of N-terminal mCherry constructs utilized (FL = full length, ΔN= deletion of N terminus, BTB = BTB domain only, C-Term = C terminus only). B, immunoprecipitation (IP) of Gβγ–Venus complexes from HEK293 cells with anti-GFP antibody followed by probing for mCherry-KCTD2 proteins with an anti-mCherry antibody. Experiments were performed in the presence of AlF4- (30 μM) in the lysis buffer. Representative blot from three independent experiments. C, IP of Gβγ–Venus complexes from HEK293 cells with anti-GFP antibody followed by probing for mCherry-KCTD5 proteins with an anti-mCherry antibody. Experiments were performed in the presence of AlF4- (30 μM) in the lysis buffer. Representative blot from three independent experiments. D, IP of Gβγ–Venus complexes from HEK293 cells with anti-GFP antibody followed by probing for mCherry-KCTD17 proteins with an anti-mCherry antibody. Experiments were performed in the presence of AlF4- (30 μM) in the lysis buffer. Representative blot from three independent experiments. E, alignment of C-terminal animo acid residues in KCTD2, 5, 17. Purple region highlights coiled-coil domain unique to KCTD17. F, scheme of constructs utilized that contain an N-terminal mCherry fused to the indicated C-terminal animo acids of KCTD17. G, IP of Gβγ–Venus complexes from HEK293 cells with anti-GFP antibody followed by probing for mCherry-KCTD17 C-terminal proteins with an anti-mCherry antibody. Experiments were performed in the presence of AlF4- (30 μM) in the lysis buffer. Representative blot from three independent experiments. HEK293, human embryonic kidney 293 cell line; KCTD, potassium channel tetramerization domain.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We next wanted to investigate downstream consequences of KCTD interaction with Gβγ in the context of intracellular GPCR signaling. For this purpose, we examined real-time cAMP dynamics by imaging the TEpacVV FRET-based biosensor in live HEK293 cells (31Klarenbeek J.B. Goedhart J. Hink M.A. Gadella T.W. Jalink K. A mTurquoise-based cAMP sensor for both FLIM and ratiometric read-out has improved dynamic range.PLoS One. 2011; 6e19170Crossref PubMed Scopus (157) Google Scholar, 32Muntean B.S. Zucca S. MacMullen C.M. Dao M.T. Johnston C. Iwamoto H. et al.Interrogating the spatiotemporal landscape of neuromodulatory GPCR signaling by real-time imaging of cAMP in intact neurons and circuits.Cell Rep. 2018; 22: 255-268Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). While numerous inputs shape adenylyl cyclase (AC)–mediated cAMP production, sensitization of AC type 5 (AC5) following prolonged Gi/o signaling involves Gβγ (33Muntean B.S. Masuho I. Dao M. Sutton L.P. Zucca S. Iwamoto H. et al.Galphao is a major determinant of cAMP signaling in the pathophysiology of movement disorders.Cell Rep. 2021; 34108718Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 34Watts V.J. Molecular mechanisms for heterologous sensitization of adenylate cyclase.J. Pharmacol. Exp. Ther. 2002; 302: 1-7Crossref PubMed Scopus (106) Google Scholar). Therefore, we first optimized conditions to robustly interrogate Gβγ influence on cAMP. For each experiment, we overexpressed D2R, AC5, and TEpacVV (Fig. 6A). Stimulation of the endogenous Gαs-coupled β2-adrenergic receptor with 1 μM isoproterenol generated a robust increase in cAMP (Fig. 6B). The response was similar in cells with overexpression of a Gi/o inhibitor (pertussis toxin S1 subunit; PTX) or a Gβγ scavenger (GRK3ct) (Fig. 6C). In these transfection conditions, KCTD (KCTD2, KCTD5, or KCTD17) overexpression also did not alter isoproterenol-induced responses compared with control cells (Fig. 6, D and E). We next induced sensitization of AC5 by stimulating D2R with 100 μM dopamine for 1 h, which resulted in an enhanced cAMP response following subsequent application of 1 μM isoproterenol (Fig. 6F). PTX blocked the sensitized response resulting in similar amplitude to non-dopamine treated cells. GRK3ct blocked sensitization in addition to generating a significantly smaller amplitude than PTX-treated cells, likely because of ongoing AC5 inhibition by D2R→Gαi signaling. Overall, the results suggest a cAMP readout dependent on Gβγ, and we therefore next assessed the impact of KCTD overexpression. In agreement with our interaction studies, we found that cAMP sensitization was blunted by KCTD2, KCTD5, and KCTD17 (Fig. 6G). Curiously, KCTD9 slightly inhibited cAMP sensitization, suggesting a potential non-Gβγ effect (Fig. 6H). We also found that the KCTD9 mutant containing charged/polar residues from KCTD2, KCTD5, and KCTD17 decreased cAMP amplitude (Fig. 6H). Finally, the KCTD17 C terminus blunted cAMP response consistent with data obtained from the GRK3ct scavenger (Fig. 6H). Overall, these experiments support a model that KCTD interaction with Gβγ sequesters AC5-mediated sensitization of cAMP. As data in the reconstituted system support a role for KCTD overexpression to uncouple Gβγ from AC5, we next w
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