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The COOH-terminal domain of huntingtin interacts with RhoGEF kalirin and modulates cell survival.

ABSTRACT: Human huntingtin (Htt) contains 3144 amino acids and has an expanded polyglutamine region near the NH2-terminus in patients with Huntington's disease. While numerous binding partners have been identified to NH2-terminal Htt, fewer proteins are known to interact with C-terminal domains of Htt. Here we report that kalirin, a Rac1 activator, is a binding partner to C-terminal Htt. Kalirin and Htt co-precipitated from mouse brain endosomes and co-localized at puncta in NRK and immortalized striatal cells and primary cortical neurons. We mapped the interaction domains to kalirin674-1272 and Htt2568-3144 and determined that the interaction between kalirin and Htt was independent of HAP1, a known interactor for Htt and kalirin. Kalirin precipitated with mutant Htt was more abundant than with wild-type Htt and had a reduced capacity to activate Rac1 when mutant Htt was present. Expression of Htt2568-3144 caused cytotoxicity, partially rescued by co-expressing kalirin674-1272 but not other regions of kalirin. Our study suggests that the interaction of kalirin with the C-terminal region of Htt influences the function of kalirin and modulates the cytotoxicity induced by C-terminal Htt.

SUBMITTER: McClory H

PROVIDER: S-EPMC5964228 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature

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The COOH-terminal domain of huntingtin interacts with RhoGEF kalirin and modulates cell survival.

McClory Hollis H Wang Xiaolong X Sapp Ellen E Gatune Leah W LW Iuliano Maria M Wu Chiu-Yi CY Nathwani Gina G Kegel-Gleason Kimberly B KB DiFiglia Marian M Li Xueyi X

Scientific reports 20180522 1

Human huntingtin (Htt) contains 3144 amino acids and has an expanded polyglutamine region near the NH<sub>2</sub>-terminus in patients with Huntington's disease. While numerous binding partners have been identified to NH<sub>2</sub>-terminal Htt, fewer proteins are known to interact with C-terminal domains of Htt. Here we report that kalirin, a Rac1 activator, is a binding partner to C-terminal Htt. Kalirin and Htt co-precipitated from mouse brain endosomes and co-localized at puncta in NRK and ...[more]

PMID: 29789657

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Project description:Kalirin (Kal), a dual Rho GDP/GTP exchange factor (GEF), plays essential roles within and outside the nervous system. Tissue-specific, developmentally regulated alternative splicing generates isoforms with one (Kal7) or two (Kal9, Kal12) GEF domains along with a kinase (Kal12) domain; while Kal9 and Kal12 are crucial for neurite outgrowth, Kal7 plays important roles in spine maintenance and synaptic plasticity. Tissue-specific usage of alternate Kalrn promoters (A, B, C, D) places four different peptides before the Sec14 domain. cSec14, with an amphipathic helix encoded by the C-promoter (Kal-C-helix), is the only variant known to interact with phosphoinositides. We sought to elucidate the biological significance of Kalirin promoter usage and lipid binding. While Ex1B expression was predominant early in development, Ex1C expression increased when synaptogenesis occurred. Kal-C-helix-containing Kal7 (cKal7) was enriched at the postsynaptic density, present in the microsomal fraction and absent from cytosol; no significant amount of cKal9 or cKal12 could be identified in mouse brain. Similarly, in primary hippocampal neurons, endogenous cKalirin colocalized with postsynaptic density 95 in dendritic spines, juxtaposed to Vglut1-positive puncta. When expressed in young neurons, bSec14-EGFP was diffusely distributed, while cSec14-EGFP localized to internal puncta. Transfected bKal7-EGFP and cKal7-EGFP localized to dendritic spines and increased spine density in more mature cultured neurons. Although promoter usage did not alter the Rac-GEF activity of Kal7, the synaptic puncta formed by cKal7-EGFP were smaller than those formed by bKal7-EGFP. Molecular modeling predicted a role for Kal-C-helix residue Arg15 in the interaction of cSec14 with phosphoinositides. Consistent with this prediction, mutation of Arg15 to Gln altered the localization of cSec14-EGFP and cKal7-EGFP. These data suggest that phosphoinositide-dependent interactions unique to cKal7 contribute to protein localization and function. Cover Image for this issue: doi. 10.1111/jnc.13791.

Project description:Magnesium levels in cardiac myocytes change in cardiovascular diseases. Intracellular free magnesium (Mg(i)) inhibits L-type Ca(2+) currents through Ca(V)1.2 channels in cardiac myocytes, but the mechanism of this effect is unknown. We hypothesized that Mg(i) acts through the COOH-terminal EF-hand of Ca(V)1.2. EF-hand mutants were engineered to have either decreased (D1546A/N/S/K) or increased (K1543D and K1539D) Mg(2+) affinity. In whole-cell patch clamp experiments, increased Mg(i) reduced both Ba(2+) and Ca(2+) currents conducted by wild type (WT) Ca(V)1.2 channels expressed in tsA-201 cells with similar affinity. Exposure of WT Ca(V)1.2 to lower Mg(i) (0.26 mM) increased the amplitudes of Ba(2+) currents 2.6 +/- 0.4-fold without effects on the voltage dependence of activation and inactivation. In contrast, increasing Mg(i) to 2.4 or 7.2 mM reduced current amplitude to 0.5 +/- 0.1 and 0.26 +/- 0.05 of the control level at 0.8 mM Mg(i). The effects of Mg(i) on peak Ba(2+) currents were approximately fit by a single binding site model with an apparent K(d) of 0.65 mM. The apparent K(d) for this effect of Mg(i) was shifted approximately 3.3- to 16.5-fold to higher concentration in D1546A/N/S mutants, with only small effects on the voltage dependence of activation and inactivation. Moreover, mutant D1546K was insensitive to Mg(i) up to 7.2 mM. In contrast to these results, peak Ba(2+) currents through the K1543D mutant were inhibited by lower concentrations of Mg(i) compared with WT, consistent with approximately fourfold reduction in apparent K(d) for Mg(i), and inhibition of mutant K1539D by Mg(i) was also increased comparably. In addition to these effects, voltage-dependent inactivation of K1543D and K1539D was incomplete at positive membrane potentials when Mg(i) was reduced to 0.26 or 0.1 mM, respectively. These results support a novel mechanism linking the COOH-terminal EF-hand with modulation of Ca(V)1.2 channels by Mg(i). Our findings expand the repertoire of modulatory interactions taking place at the COOH terminus of Ca(V)1.2 channels, and reveal a potentially important role of Mg(i) binding to the COOH-terminal EF-hand in regulating Ca(2+) influx in physiological and pathophysiological states.

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The COOH-terminal domain of huntingtin interacts with RhoGEF kalirin and modulates cell survival.

Publications

The COOH-terminal domain of huntingtin interacts with RhoGEF kalirin and modulates cell survival.

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