Alternate promoter usage generates two subpopulations of the neuronal RhoGEF Kalirin-7

Abstract

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.

Keywords: Sec14; amphipathic helix; dendritic spine; phosphoinositide; postsynaptic density; synaptogenesis.