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Alternate promoter usage generates two subpopulations of the neuronal Rho GEF Kalirin‐7
Author(s) -
Miller Megan B.,
Yan Yan,
Wu Yi,
Hao Bing,
Mains Richard E.,
Eipper Betty A.
Publication year - 2017
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/jnc.13749
Subject(s) - synaptogenesis , dendritic spine , neurite , microbiology and biotechnology , postsynaptic density , postsynaptic potential , chemistry , guanine nucleotide exchange factor , gene isoform , biology , cytosol , hippocampal formation , biochemistry , gtpase , neuroscience , gene , receptor , in vitro , enzyme
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. cS ec14, 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 ( cK al7) was enriched at the postsynaptic density, present in the microsomal fraction and absent from cytosol; no significant amount of cK al9 or cK al12 could be identified in mouse brain. Similarly, in primary hippocampal neurons, endogenous cK alirin colocalized with postsynaptic density 95 in dendritic spines, juxtaposed to Vglut1‐positive puncta. When expressed in young neurons, bS ec14‐ EGFP was diffusely distributed, while cS ec14‐ EGFP localized to internal puncta. Transfected bK al7‐ EGFP and cK al7‐ 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 cK al7‐ EGFP were smaller than those formed by bK al7‐ EGFP . Molecular modeling predicted a role for Kal‐C‐helix residue Arg 15 in the interaction of cS ec14 with phosphoinositides. Consistent with this prediction, mutation of Arg 15 to Gln altered the localization of cS ec14‐ EGFP and cK al7‐ EGFP . These data suggest that phosphoinositide‐dependent interactions unique to cK al7 contribute to protein localization and function.Cover Image for this issue: doi. 10.1111/jnc.13791.