Members of the KCTD family are major regulators of cAMP signaling

Abstract

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.

Keywords: GPCR; cAMP; neuron; striatum; zinc.

Fig. 1.

KCTD proteins differentially regulate cAMP signaling in striatal neurons. (A) Scheme of inputs to AC5 in striatal medium spiny neurons. (B) CRISPR/Cas9 gene editing strategy in CAMPER primary striatal neurons. (C) Western blot for KCTD level in primary striatal neurons subject to CRISPR/Cas9 targeting of KCTDs or control. n = 4 cultures for each sgRNA. (D) Baseline cAMP values in D1R+ and D2R+ striatal neurons classified by directionality of response to dopamine. n ≥ 9 neurons/group; one-way ANOVA; and #P < 0.0001. (E) Averaged cAMP responses to bath application of 100 μM forskolin. n ≥ 16 neurons/group. (F) Max cAMP response amplitude to 100 μM forskolin in striatal neurons. n ≥ 16 neurons/group; one-way ANOVA; and ****P < 0.0001. (G) Dopamine → D1R dose–response curve. n ≥ 13 neurons/dose. (H) Max D1R response amplitude from 100 μM dopamine. n ≥ 10 neurons/group; one-way ANOVA; ***P < 0.001; and ****P < 0.0001. (I) D1R response EC₅₀ from dopamine. n ≥ 13 neurons/dose; one-way ANOVA; and ****P < 0.0001. (J) Dopamine → D2R dose–response curve. n ≥ 9 neurons/dose. (K) Max D2R response amplitude from 100 μM dopamine. n ≥ 9 neurons/group. (L) D2R response EC₅₀ from dopamine. n ≥ 9 neurons/dose. All data represented as mean ± SEM.

Fig. 2.

KCTD5 modulates AC5 activity through Zip14-mediated zinc flux. (A) Averaged cAMP responses to bath application of 100 μM forskolin in striatal neurons in the presence or absence of zinc chloride (100 μM). n = 16 neurons/group. (B) Max cAMP response amplitude to 100 μM forskolin in the presence or absence of zinc chloride (100 μM). n = 16 neurons/group; nonparametric Mann–Whitney U test; and ****P < 0.0001. (C) Western blot for Zip14 level in primary striatal neurons subject to CRISPR/Cas9 targeting of KCTDs or control. n = 4 cultures/group; one-way ANOVA Tukey comparison test to control sgRNA; *P < 0.05; and **P < 0.01. (D) Western blot from HEK293 cells with indicated transfection and 2-h treatment with dimethyl sulfoxide (DMSO) or Bortezomib (100 nM). Bands labeled for ubiquitinated Zip14-Ub and Zip14-DG. Representative blot of three independent experiments. (E) Scheme of Fluozin-3 sensor responding to Zn²⁺. (F) Averaged Zn2+ responses to bath application of 1 μM zinc chloride in striatal neurons (n = 27 control CRISPR/Cas9, n = 25 KCTD5 CRISPR/Cas9 knockout, and n = 15 Zip14 overexpression) and rate of Zn2+ flux in striatal neurons. n = 27 control CRISPR/Cas9 (r² = 0.969), n = 25 KCTD5 CRISPR/Cas9 knockout (r² = 0.967), n = 15 Zip14 overexpression (r² = 0.979); one-way ANOVA Dunnett’s multiple comparison; and ****P < 0.0001. (G) Zinc flux dose–response curve. n ≥ 9 neurons/dose. (H) Zinc flux EC₅₀. n ≥ 9 neurons/dose; one-way ANOVA Dunnett’s multiple comparison; and **P < 0.01. (I) Max zinc flux. n ≥ 11neurons/group; one-way ANOVA Dunnett’s multiple comparison; *P < 0.05, and ****P < 0.0001. (J) Averaged cAMP responses to bath application of 100 μM forskolin. n ≥ 9 neurons/group. (K) Max cAMP response amplitude to 100 μM forskolin in striatal neurons. n ≥ 9 neurons/group; one-way ANOVA Dunnett’s multiple comparison; and ****P < 0.0001. (L) AC5 and Zip14 coimmunoprecipitate in transfected HEK293T/17 cells. Representative data from three independent experiments. All data represented as mean ± SEM.

Fig. 3.

KCTD5 sensitizes striatal cAMP signaling through Gβγ regulation. (A) Western blot analysis from cultured striatal neurons subject to CRISPR/Cas9 targeting of either KCTD5 or scrambled control. n = 6 cultures/group; nonparametric Mann–Whitney U test; *P < 0.05; and **P < 0.01. (B) Western blot from HEK293 cell with indicated transfection (Gαo, Gβ2, Gγ7, ubiquitin, and KCTD5) and 2-h treatment with AlF₄ (30 μM), Bortezomib (100 nM), or both. Band labeled for ubiquitinated Gβ (Gβ-Ub). Representative blot of three independent experiments. (C) Gβγ-mediated sensitization of Gαolf-AC-cAMP signaling is attenuated by the Gβγ-scavenger Grk3ct. (D) Representative D1R-mediated cAMP responses to a phasic puff of 100 μM dopamine (Left), with Grk3ct overexpression (Gβγ block; Middle) or Gβγ overexpression (+Gβγ; Right). n ≥ 8 neurons/group. (E) Max D1R response amplitude from 100 μM dopamine. n ≥ 8 neurons/group; nonparametric Mann–Whitney U test; and ****P < 0.0001. (F) Dopamine → D1R dose–response curve with Grk3ct overexpression (Gβγ block; Left) or Gβγ overexpression (+Gβγ; Right). n ≥ 8 neurons/dose. (G) D1R response EC₅₀ from dopamine. n ≥ 14 neurons/dose; nonparametric Mann–Whitney U test; ns P > 0.05; ***P < 0.001; and ****P < 0.0001. All data represented as mean ± SEM.

Fig. 4.

KCTD5 haploinsufficiency drives motor impairments in mice. A total of 20 mice in two groups of Kctd5 mice (i.e., wild-type [Kctd5^+/+], n = 6 [male] and 4 [female], and heterozygous [Kctd5^+/−], n = 5 [male] and 5 [female]) were used for behavioral analysis. (A) Western blot from dorsal striatal tissue punches taken from Kctd5^+/+ and Kctd5^+/−. n = 10 mice per group; nonparametric Mann–Whitney U test; **P = 0.0052; and ***P = 0.0002. (B) Hindlimb-clasping pathology score for Kctd5^+/+ (n = 10) and Kctd5^+/− (n = 10) mice (unpaired t test; parametric; and **P = 0.003). (C) Ledge test pathology score for Kctd5^+/+ (n = 10) and Kctd5^+/− (n = 10) mice (unpaired t test; parametric; and **P = 0.003). (D) Horizontal pole test for Kctd5^+/+ (n = 10) and Kctd5^+/− (n = 10) mice with varying pole diameter: 15.8 (L), 11 (M), and 8 (S) mm (Left). Time to complete test is shown (two-way ANOVA; Sidak multiple comparison test; and ***P < 0.001) (Right). Number of hindlimb slips during test (two-way ANOVA; Sidak multiple comparison test; and ***P < 0.001). (E) Vertical pole test score for Kctd5^+/+ (n = 10) and Kctd5^+/− (n = 10) mice (unpaired t test; parametric; and ***P < 0.001). (F) Learning rate on accelerating rotarod for Kctd5^+/+ (n = 10) and Kctd5^+/− (n = 10) mice. (Two-way ANOVA; Sidak multiple comparison test; and *P = 0.038). (G) Latency to fall while walking backward on a rotating beam for Kctd5^+/+ (n = 10) and Kctd5^+/− (n = 10) mice (Left). Three intraday trials over 3 d are shown (Right). Average of daily latency (two-way ANOVA; Sidak multiple comparison test; **P = 0.003, and ***P < 0.001). (H) Hindlimb-clasping pathology score for mice treated with vehicle (Kctd5^+/+ n = 4, Kctd5^+/− n = 5) or TPEN (Kctd5^+/+ n = 6, Kctd5^+/− n = 5). Two-way ANOVA; Sidak multiple comparison test; and *P = 0.0295. (I) Latency to fall while walking backward on a rotating beam for mice treated with vehicle (Kctd5^+/+ n = 4, Kctd5^+/− n = 5) or TPEN (Kctd5^+/+ n = 6, Kctd5^+/− n = 5) (Left). Average from interday trials; two-way repeated measures (RM) ANOVA; Tukey multiple comparison test; and *P < 0.05 for Kctd5^+/− (TPEN) versus other groups (Right). Average latency from all trials; two-way ANOVA; Sidak multiple comparison test; and **P = 0.004. (J) Vertical pole test score for mice treated with vehicle (Kctd5^+/+ n = 4, Kctd5^+/− n = 5) or TPEN (Kctd5^+/+ n = 6, Kctd5^+/− n = 5). Two-way ANOVA; Sidak multiple comparison test; and ***P = 0.0004. (K) Ledge test pathology score for mice treated with vehicle (Kctd5^+/+ n = 4, Kctd5^+/− n = 5) or TPEN (Kctd5^+/+ n = 6, Kctd5^+/− n = 5). Two-way ANOVA; Sidak multiple comparison test. All data represented as mean ± SEM.

Fig. 5.

Dual modulation of AC5 activity by KCTD mediated through Gβγ and Zip14. Schematic illustration of distinct binding domains on AC5. Occupation of binding sites enable substrates to modulate degree of open (active) and closed (inactive) conformation state of the enzyme, subsequently altering catalytic activity. Green substrates favor open (active) state, and red substrates favor closed (inactive) state. Gβγ binds intracellular N terminus. Gαi and Gαolf have distinct binding site on catalytic domains (C1 and C2, respectively). Forskolin (FSK) and Zn²⁺ bind near ATP. In our model, Zip14 proximity facilitates loading Zn²⁺ in AC5. KCTD5 inhibits both Gβγ and Zip14, thus exerting dual regulation over AC5 activity.