KCTD Proteins Research Articles

KCTD proteins regulate morphine dependence via heterologous sensitization of adenylyl cyclase 1 in mice.

Heterologous sensitization of adenylyl cyclase (AC) results in elevated cAMP signaling transduction that contributes to drug dependence. Inhibiting cullin3-RING ligases by blocking the neddylation of cullin3 abolishes heterologous sensitization, however, the modulating mechanism remains uncharted. Here, we report an essential role of the potassium channel tetramerization domain (KCTD) protein 2, 5, and 17, especially the dominant isoform KCTD5 in regulating heterologous sensitization of AC1 and morphine dependence via working with cullin3 and the cullin-associated and neddylation-dissociated 1 (CAND1) protein. In cellular models, we observed enhanced association of KCTD5 with Gβ and cullin3, along with elevated dissociation of Gβ from AC1 as well as of CAND1 from cullin3 in heterologous sensitization of AC1. Given binding of CAND1 inhibits the neddylation of cullin3, we further elucidated that the enhanced interaction of KCTD5 with both Gβ and cullin3 promoted the dissociation of CAND1 from cullin3, attenuated the inhibitory effect of CAND1 on cullin3 neddylation, ultimately resulted in heterologous sensitization of AC1. The paraventricular thalamic nucleus (PVT) plays an important role in mediating morphine dependence. Through pharmacological and biochemical approaches, we then demonstrated that KCTD5/cullin3 regulates morphine dependence via modulating heterologous sensitization of AC, likely AC1 in PVT in mice. In summary, the present study revealed the underlying mechanism of heterologous sensitization of AC1 mediated by cullin3 and discovered the role of KCTD proteins in regulating morphine dependence in mice.

KCTD Proteins Have Redundant Functions in Controlling Cellular Growth.

We explored the functional redundancy of three structurally related KCTD (Potassium Channel Tetramerization Domain) proteins, KCTD2, KCTD5, and KCTD17, by progressively knocking them out in HEK 293 cells using CRISPR/Cas9 genome editing. After validating the knockout, we assessed the effects of progressive knockout on cell growth and gene expression. We noted that the progressive effects of knockout of KCTD isoforms on cell growth were most pervasive when all three isoforms were deleted, suggesting some functions were conserved between them. This was also reflected in progressive changes in gene expression. Our previous work indicated that Gβ1 was involved in the transcriptional control of gene expression, so we compared the gene expression patterns between GNB1 and KCTD KO. Knockout of GNB1 led to numerous changes in the expression levels of other G protein subunit genes, while knockout of KCTD isoforms had the opposite effect, presumably because of their role in regulating levels of Gβ1. Our work demonstrates a unique relationship between KCTD proteins and Gβ1 and a global role for this subfamily of KCTD proteins in maintaining the ability of cells to survive and proliferate.

Preassembly of specific Gβγ subunits at GABAB receptors through auxiliary KCTD proteins accelerates channel gating

GABAB receptors (GBRs) are G protein-coupled receptors for GABA, the main inhibitory neurotransmitter in the brain. GBRs regulate fast synaptic transmission by gating Ca2+ and K+ channels via the Gβγ subunits of the activated G protein. It has been demonstrated that auxiliary GBR subunits, the KCTD proteins, shorten onset and rise time and increase desensitization of receptor-induced K+ currents. KCTD proteins increase desensitization of K+ currents by scavenging Gβγ from the channel, yet the mechanism responsible for the rapid activation of K+ currents has remained elusive. In this study, we demonstrate that KCTD proteins preassemble Gβγ at GBRs. The preassembly obviates the need for diffusion-limited G protein recruitment to the receptor, thereby accelerating G protein activation and, as a result, K+ channel activation. Preassembly of Gβγ at the receptor relies on the interaction of KCTD proteins with a loop protruding from the seven-bladed propeller of Gβ subunits. The binding site is shared between Gβ1 and Gβ2, limiting the interaction of KCTD proteins to these particular Gβ isoforms. Substituting residues in the KCTD binding site of Gβ1 with those from Gβ3 hinders the preassembly of Gβγ with GBRs, delays onset and prolongs rise time of receptor-activated K+ currents. The KCTD-Gβ interface, therefore, represents a target for pharmacological modulation of channel gating by GBRs.

A Comprehensive Analysis of the Structural Recognition between KCTD Proteins and Cullin 3.

KCTD ((K)potassium Channel Tetramerization Domain-containing) proteins constitute an emerging class of proteins involved in fundamental physio-pathological processes. In these proteins, the BTB domain, which represents the defining element of the family, may have the dual role of promoting oligomerization and favoring functionally important partnerships with different interactors. Here, by exploiting the potential of recently developed methodologies for protein structure prediction, we report a comprehensive analysis of the interactions of all KCTD proteins with their most common partner Cullin 3 (Cul3). The data here presented demonstrate the impressive ability of this approach to discriminate between KCTDs that interact with Cul3 and those that do not. Indeed, reliable and stable models of the complexes were only obtained for the 15 members of the family that are known to interact with Cul3. The generation of three-dimensional models for all KCTD-Cul3 complexes provides interesting clues on the determinants of the structural basis of this partnership as clear structural differences emerged between KCTDs that bind or do not bind Cul3. Finally, the availability of accurate three-dimensional models for KCTD-Cul3 interactions may be valuable for the ad hoc design and development of compounds targeting specific KCTDs that are involved in several common diseases.

KCTD5 Forms Hetero-Oligomeric Complexes with Various Members of the KCTD Protein Family.

Potassium Channel Tetramerization Domain 5 (KCTD5) regulates diverse aspects of physiology, ranging from neuronal signaling to colorectal cancer. A key feature of KCTD5 is its self-assembly into multi-subunit oligomers that seemingly enables participation in an array of protein-protein interactions. KCTD5 has recently been reported to form hetero-oligomeric complexes with two similar KCTDs (KCTD2 and KCTD17). However, it is not known if KCTD5 forms hetero-oligomeric complexes with the remaining KCTD protein family which contains over two dozen members. Here, we demonstrate that KCTD5 interacts with various KCTD proteins when assayed through co-immunoprecipitation in lysed cells. We reinforced this dataset by examining KCTD5 interactions in a live-cell bioluminescence resonance energy transfer (BRET)-based approach. Finally, we developed an IP-luminescence approach to map regions on KCTD5 required for interaction with a selection of KCTD that have established roles in neuronal signaling. We report that different regions on KCTD5 are responsible for uniquely contributing to interactions with other KCTD proteins. While our results help unravel additional interaction partners for KCTD5, they also reveal additional complexities in KCTDs' biology. Moreover, our findings also suggest that KCTD hetero-oligomeric interactions may occur throughout the KCTD family.

A Comprehensive Analysis of the Expression Profiles of KCTD Proteins in Acute Lymphoblastic Leukemia: Evidence of Selective Expression of KCTD1 in T-ALL.

Acute leukemia is the most common pediatric cancer. In most cases, this disease results from the malignant transformation of either the B-cell (B-ALL) or, less frequently, T-cell progenitors (T-ALL). Recently, a marked overexpression of KCTD15, a member of the emerging class of the potassium (K) channel tetramerization domain-containing proteins (KCTDs) has been detected in both patients and continuous cell lines as in vitro model systems. Because there is growing evidence of the key, yet diversified, roles played by KCTDs in cancers, we here report an exhaustive analysis of their expression profiles in both B-ALL and T-ALL patients. Although for most KCTDs, no significant alterations were found in these pathological states, for some members of the family, significant up- and down-regulations were detected in comparison with the values found in healthy subjects in the transcriptome analysis. Among these, particularly relevant is the upregulation of the closely related KCTD1 and KCTD15 in T-ALL patients. Interestingly, KCTD1 is barely expressed in both unaffected controls and B-ALL patients. Therefore, not only does this analysis represent the first study in which the dysregulation of all KCTDs is simultaneously evaluated in specific pathological contexts, but it also provides a promising T-ALL biomarker that could be suitable for clinical applications.

The Oncosuppressive Properties of KCTD1: Its Role in Cell Growth and Mobility.

The KCTD protein family is traditionally regarded as proteins that play key roles in neurological physiopathology. However, new studies are increasingly demonstrating their involvement in many other biological processes, including cancers. This is particularly evident for KCTD proteins not involved in protein ubiquitination and degradation, such as KCTD1. We explored the role of KCTD1 in colorectal cancer by knocking down this protein in the human colon adenocarcinoma cell line, SW480. We re-assessed its ability to downregulate β-catenin, a central actor in the WNT/β-catenin signalling pathway. Interestingly, opposite effects are observed when the protein is upregulated in CACO2 colorectal cancer cells. Moreover, interrogation of the TCGA database indicates that KCTD1 downregulation is associated with β-catenin overexpression in colorectal cancer patients. Indeed, knocking down KCTD1 in SW480 cells led to a significant increase in their motility and stemness, two important tumorigenesis traits, suggesting an oncosuppressor role for KCTD1. It is worth noting that similar effects are induced on colorectal cancer cells by the misregulation of KCTD12, a protein that is distantly related to KCTD1. The presented results further expand the spectrum of KCTD1 involvement in apparently unrelated physiopathological processes. The similar effects produced on colorectal cancer cell lines by KCTD1 and KCTD12 suggest novel, previously unreported analogous activities among members of the KCTD protein family.

Multiple potassium channel tetramerization domain (KCTD) family members interact with Gβγ, with effects on cAMP signaling

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.

Members of the KCTD family are major regulators of cAMP signaling

Cyclic adenosine monophosphate (cAMP) is a pivotal second messenger with an essential role in neuronal function. cAMP synthesis by adenylyl cyclases (AC) is controlled by G protein-coupled receptor (GPCR) signaling systems. However, the network of molecular players involved in the process is incompletely defined. Here, we used CRISPR/Cas9-based screening to identify that members of the potassium channel tetradimerization domain (KCTD) family are major regulators of cAMP signaling. Focusing on striatal neurons, we show that the dominant isoform KCTD5 exerts its effects through an unusual mechanism that modulates the influx of Zn2+ via the Zip14 transporter to exert unique allosteric effects on AC. We further show that KCTD5 controls the amplitude and sensitivity of stimulatory GPCR inputs to cAMP production by Gβγ-mediated AC regulation. Finally, we report that KCTD5 haploinsufficiency in mice leads to motor deficits that can be reversed by chelating Zn2+ Together, our findings uncover KCTD proteins as major regulators of neuronal cAMP signaling via diverse mechanisms.

AlphaFold-Predicted Structures of KCTD Proteins Unravel Previously Undetected Relationships among the Members of the Family.

One of the most striking features of KCTD proteins is their involvement in apparently unrelated yet fundamental physio-pathological processes. Unfortunately, comprehensive structure–function relationships for this protein family have been hampered by the scarcity of the structural data available. This scenario is rapidly changing due to the release of the protein three-dimensional models predicted by AlphaFold (AF). Here, we exploited the structural information contained in the AF database to gain insights into the relationships among the members of the KCTD family with the aim of facilitating the definition of the structural and molecular basis of key roles that these proteins play in many biological processes. The most important finding that emerged from this investigation is the discovery that, in addition to the BTB domain, the vast majority of these proteins also share a structurally similar domain in the C-terminal region despite the absence of general sequence similarities detectable in this region. Using this domain as reference, we generated a novel and comprehensive structure-based pseudo-phylogenetic tree that unraveled previously undetected similarities among the protein family. In particular, we generated a new clustering of the KCTD proteins that will represent a solid ground for interpreting their many functions.

KCTD15 deregulation is associated with alterations of the NF-\u03baB signaling in both pathological and physiological model systems

Like other KCTD proteins, KCTD15 is involved in important albeit distinct biological processes as cancer, neural crest formation, and obesity. Here, we characterized the role of KCTD15 in different physiological/pathological states to gain insights into its diversified function(s). The silencing of KCTD15 in MLL-rearranged leukemia models induced attenuation of the NF-κB pathway associated with a downregulation of pIKK-β and pIKB-α. Conversely, the activation of peripheral blood T cells upon PMA/ionomycin stimulation remarkably upregulated KCTD15 and, simultaneously, pIKK-β and pIKB-α. Moreover, a significant upregulation of KCTD15 was also observed in CD34 hematopoietic stem/progenitor cells where the NF-κB pathway is physiologically activated. The association between KCTD15 upregulation and increased NF-κB signaling was confirmed by luciferase assay as well as KCTD15 and IKK-β proximity ligation and immunoprecipitation experiments. The observed upregulation of IKK-β by KCTD15 provides a novel and intriguing interpretative key for understanding the protein function in a wide class of physiological/pathological conditions ranging from neuronal development to cancer and obesity/diabetes.

Human Multisubunit E3 Ubiquitin Ligase Required for Heterotrimeric G-Protein β-Subunit Ubiquitination and Downstream Signaling.

G-protein-coupled receptors (GPCRs) initiate intracellular signaling events through heterotrimeric G-protein α-subunits (Gα) and the βγ-subunit dimer (Gβγ). In this study, we utilized mass spectrometry to identify novel regulators of Gβγ signaling in human cells. This prompted our characterization of KCTD2 and KCTD5, two related potassium channel tetramerization domain (KCTD) proteins that specifically recognize Gβγ. We demonstrated that these KCTD proteins are substrate adaptors for a multisubunit CUL3-RING ubiquitin ligase, in which a KCTD2-KCTD5 hetero-oligomer associates with CUL3 through KCTD5 subunits and recruits Gβγ through both KCTD proteins in response to G-protein activation. These KCTD proteins promote monoubiquitination of lysine-23 within Gβ1/2in vitro and in HEK-293 cells. Depletion of these adaptors from cancer cell lines sharply impairs downstream signaling. Together, our studies suggest that a KCTD2-KCTD5-CUL3-RING E3 ligase recruits Gβγ in response to signaling, monoubiquitinates lysine-23 within Gβ1/2, and regulates Gβγ effectors to modulate downstream signal transduction.

The Structural Versatility of the BTB Domains of KCTD Proteins and Their Recognition of the GABAB Receptor.

Several recent investigations have demonstrated that members of the KCTD (Potassium Channel Tetramerization Domain) protein family are involved in fundamental processes. However, the paucity of structural data available on these proteins has frequently prevented the definition of their biochemical role(s). Fortunately, this scenario is rapidly changing as, in very recent years, several crystallographic structures have been reported. Although these investigations have provided very important insights into the function of KCTDs, they have also raised some puzzling issues. One is related to the observation that the BTB (broad-complex, tramtrack, and bric-à-brac) domain of these proteins presents a remarkable structural versatility, being able to adopt a variety of oligomeric states. To gain insights into this intriguing aspect, we performed extensive molecular dynamics simulations on several BTB domains of KCTD proteins in different oligomeric states (monomers, dimers, tetramers, and open/close pentamers). These studies indicate that KCTD-BTB domains are stable in the simulation timescales, even in their monomeric forms. Moreover, simulations also show that the dynamic behavior of open pentameric states is strictly related to their functional roles and that different KCTDs may form stable hetero-oligomers. Molecular dynamics (MD) simulations also provided a dynamic view of the complex formed by KCTD16 and the GABAB2 receptor, whose structure has been recently reported. Finally, simulations carried out on the isolated fragment of the GABAB2 receptor that binds KCTD16 indicate that it is able to assume the local conformation required for the binding to KCTD.

Structural basis for KCTD-mediated rapid desensitization of GABAB signalling.

The GABAB receptor is one of the principal inhibitory neurotransmitter receptors in the brain, and it signals through heterotrimeric G proteins to activate a variety of effectors including G protein-coupled inwardly-rectifying potassium channels (GIRKs)1,2. GABAB receptor signaling is tightly regulated by auxiliary subunits called KCTDs, which control the kinetics of GIRK activation and desensitization3–5. However, the mechanistic basis for KCTD modulation of GABAB signaling remains incompletely understood. Here, using a combination of X-ray crystallography, electron microscopy, functional and biochemical experiments we reveal the molecular details of KCTD binding to both GABAB receptors and Gβγ subunits. KCTDs associate with the receptor by forming an asymmetric pentameric ring around a region of the receptor C-terminal tail, while a second KCTD domain, H1, engages in a symmetric interaction with five copies of Gβγ in which the G protein subunits also directly interact with one another. We further show that KCTD binding to Gβγ is highly cooperative, defining a model in which KCTDs cooperatively strip G proteins from GIRK channels to induce rapid desensitization following receptor activation. These results provide a framework for understanding the molecular basis for the precise temporal control of GABAB signaling by KCTD proteins.

Structural complexity in the KCTD family of Cullin3-dependent E3 ubiquitin ligases.

Members of the potassium channel tetramerization domain (KCTD) family are soluble non-channel proteins that commonly function as Cullin3 (Cul3)-dependent E3 ligases. Solution studies of the N-terminal BTB domain have suggested that some KCTD family members may tetramerize similarly to the homologous tetramerization domain (T1) of the voltage-gated potassium (Kv) channels. However, available structures of KCTD1, KCTD5 and KCTD9 have demonstrated instead pentameric assemblies. To explore other phylogenetic clades within the KCTD family, we determined the crystal structures of the BTB domains of a further five human KCTD proteins revealing a rich variety of oligomerization architectures, including monomer (SHKBP1), a novel two-fold symmetric tetramer (KCTD10 and KCTD13), open pentamer (KCTD16) and closed pentamer (KCTD17). While these diverse geometries were confirmed by small-angle X-ray scattering (SAXS), only the pentameric forms were stable upon size-exclusion chromatography. With the exception of KCTD16, all proteins bound to Cul3 and were observed to reassemble in solution as 5 : 5 heterodecamers. SAXS data and structural modelling indicate that Cul3 may stabilize closed BTB pentamers by binding across their BTB–BTB interfaces. These extra interactions likely also allow KCTD proteins to bind Cul3 without the expected 3-box motif. Overall, these studies reveal the KCTD family BTB domain to be a highly versatile scaffold compatible with a range of oligomeric assemblies and geometries. This observed interface plasticity may support functional changes in regulation of this unusual E3 ligase family.

KCTD Hetero-oligomers Confer Unique Kinetic Properties on Hippocampal GABABReceptor-Induced K+Currents

The KCTD proteins 8, 12, and 16 are auxiliary subunits of GABAB receptors that differentially regulate G-protein signaling of the receptor. The KCTD proteins are generally assumed to function as homo-oligomers. Here we show that the KCTD proteins also assemble hetero-oligomers in all possible dual combinations. Experiments in live cells demonstrate that KCTD hetero-oligomers form at least tetramers and that these tetramers directly interact with the receptor and the G-protein. KCTD12/KCTD16 hetero-oligomers impart unique kinetic properties to GABAB receptor-induced Kir3 currents in heterologous cells. KCTD12/KCTD16 hetero-oligomers are abundant in the hippocampus, where they prolong the duration of slow IPSCs in pyramidal cells. Our data therefore support that KCTD hetero-oligomers modulate physiologically induced K+ current responses in the brain.

Bivalent Copper Ions Promote Fibrillar Aggregation of KCTD1 and Induce Cytotoxicity.

Potassium channel tetramerization domain containing 1 (KCTD1) family members have a BTB/POZ domain, which can facilitate protein-protein interactions involved in the regulation of different signaling pathways. KCTD proteins have potential Zn2+/Cu2+ binding sites with currently unknown structural and functional roles. We investigated potential Cu2+-specific effects on KCTD1 using circular dichroism, turbidity measurement, fluorescent dye binding, proteinase K (PK) digestion, cell proliferation and apoptosis assays. These experiments indicate that the KCTD1 secondary structure assumes greater β-sheet content and the proteins aggregate into a PK-resistant form under 20 μM Cu2+, and this β-sheet-rich aggregation with Cu2+ promotes fibril formation, which results in increased cell toxicity by apoptosis. Our results reveal a novel role for Cu2+ in determining the structure and function of KCTD1.

Modular composition and dynamics of native GABAB receptors identified by high-resolution proteomics

GABAB receptors, the most abundant inhibitory G protein-coupled receptors in the mammalian brain, display pronounced diversity in functional properties, cellular signaling and subcellular distribution. We used high-resolution functional proteomics to identify the building blocks of these receptors in the rodent brain. Our analyses revealed that native GABAB receptors are macromolecular complexes with defined architecture, but marked diversity in subunit composition: the receptor core is assembled from GABAB1a/b, GABAB2, four KCTD proteins and a distinct set of G-protein subunits, whereas the receptor's periphery is mostly formed by transmembrane proteins of different classes. In particular, the periphery-forming constituents include signaling effectors, such as Cav2 and HCN channels, and the proteins AJAP1 and amyloid-β A4, both of which tightly associate with the sushi domains of GABAB1a. Our results unravel the molecular diversity of GABAB receptors and their postnatal assembly dynamics and provide a roadmap for studying the cellular signaling of this inhibitory neurotransmitter receptor.

Structural Insights into KCTD Protein Assembly and Cullin3 Recognition

Cullin3 (Cul3)-based ubiquitin E3 ligase complexes catalyze the transfer of ubiquitin from an E2 enzyme to target substrate proteins. In these assemblies, the C-terminal region of Cul3 binds Rbx1/E2-ubiquitin, while the N-terminal region interacts with various BTB (bric-à-brac, tramtrack, broad complex) domain proteins that serve as substrate adaptors. Previous crystal structures of the homodimeric BTB proteins KLHL3, KLHL11 and SPOP in complex with the N-terminal domain of Cul3 revealed the features required for Cul3 recognition in these proteins. A second class of BTB-domain-containing proteins, the KCTD proteins, is also Cul3 substrate adaptors, but these do not share many of the previously identified determinants for Cul3 binding. We report the pentameric crystal structures of the KCTD1 and KCTD9 BTB domains and identify plasticity in the KCTD1 rings. We find that the KCTD proteins 5, 6, 9 and 17 bind to Cul3 with high affinity, while the KCTD proteins 1 and 16 do not have detectable binding. Finally, we confirm the 5:5 assembly of KCTD9/Cul3 complexes by cryo-electron microscopy and provide a molecular rationale for BTB-mediated Cul3 binding specificity in the KCTD family.

Cullin 3 Recognition Is Not a Universal Property among KCTD Proteins.

Cullin 3 (Cul3) recognition by BTB domains is a key process in protein ubiquitination. Among Cul3 binders, a great attention is currently devoted to KCTD proteins, which are implicated in fundamental biological processes. On the basis of the high similarity of BTB domains of these proteins, it has been suggested that the ability to bind Cul3 could be a general property among all KCTDs. In order to gain new insights into KCTD functionality, we here evaluated and/or quantified the binding of Cul3 to the BTB of KCTD proteins, which are known to be involved either in cullin-independent (KCTD12 and KCTD15) or in cullin-mediated (KCTD6 and KCTD11) activities. Our data indicate that KCTD6BTB and KCTD11BTB bind Cul3 with high affinity forming stable complexes with 4:4 stoichiometries. Conversely, KCTD12BTB and KCTD15BTB do not interact with Cul3, despite the high level of sequence identity with the BTB domains of cullin binding KCTDs. Intriguingly, comparative sequence analyses indicate that the capability of KCTD proteins to recognize Cul3 has been lost more than once in distinct events along the evolution. Present findings also provide interesting clues on the structural determinants of Cul3-KCTD recognition. Indeed, the characterization of a chimeric variant of KCTD11 demonstrates that the swapping of α2β3 loop between KCTD11BTB and KCTD12BTB is sufficient to abolish the ability of KCTD11BTB to bind Cul3. Finally, present findings, along with previous literature data, provide a virtually complete coverage of Cul3 binding ability of the members of the entire KCTD family.