Subtle functional defects in the Arf-specific guanine nucleotide exchange factor IQSEC2 cause non-syndromic X-linked intellectual disability

Abstract

Mutations in IQSEC2, a guanine nucleotide exchange factor for the ADP-ribosylation factor (Arf) family of small GTPases have recently been shown to cause non-syndromic X-linked intellectual disability (ID), characterised by substantial limitations in intellectual functioning and adaptive behaviour. This discovery was revealed by a combination of large-scale resequencing of the X chromosome, and key functional assays that revealed a reduction, but not elimination, of IQSEC2 GEF activity for mutations affecting conserved amino acids in the IQ-like and Sec7 domains. Compromised GTP binding activity of IQSEC2 leading to reduced activation of selected Arf substrates (Arf1, Arf6) is expected to impact on cytoskeletal organization, dendritic spine morphology and synaptic organisation. This study highlights the need for further investigation of the IQSEC gene family and Arf GTPases in neuronal morphology and synaptic function, and suggests that the genes encoding the ArfGEFs IQSEC1 and IQSEC3 should be considered as candidates for screening in autosomal ID.

This article refers to:

Acknowledgements

The authors acknowledge the support of the Australian NHMRC (J.G. and C.S.), the SMILE Foundation (J.G.), the Women's and Children's Hospital Foundation (C.S.), D. Harwood (J.G.) and the Medical Research Council (R.J.H.).

Figures and Tables

Figure 1 Role of Arf6 in neuronal development and synaptic plasticity. Small GTPases of the ADP ribosylation factor (Arf) family play a crucial role in the regulation of vesicular transport, organelle structure, lipid modification, membrane trafficking and actin dynamics.26 In mammals there are six ubiquitously expressed genes/paralogs, divided into three classes based on sequence similarity (Class I: Arf1–3, Class II: Arf4–5, Class III: Arf6). Class I and II Arfs are localised to the Golgi and endosomal compartments. By contrast, Arf6 is localised to cell periphery in association with the plasma membrane and a subset of endosomes.27 The exchange of GDP for GTP on Arfs is catalysed by guanine nucleotide exchange factors (GEFs). There are at least fifteen ArfGEFs in the human genome,28 each characterised by a catalytic Sec7 domain with sequence identity to the yeast ArfGEF sec7p. Active Arf6 (Arf6-GTP) activates several downstream effectors such as phospholipase D (PLD), phosphatidylinositol-4-phosphate 5-kinases (PIP5K) and Rac1, leading to changes in membrane trafficking and actin dynamics. PA, phosphatidic acid; PIP2, phosphatidylinositol bisphosphate. Modified from reference 29.

Figure 2 IQSEC2 mutations in XLID. (A) Schematic of the human IQSEC2 protein with a regulatory IQ-like motif, a catalytic Sec7 domain, a pleckstrin homology (PH) domain and a PDZ binding motif. The relative locations of mutations found in each family are shown. In the MRX 1 family (MIM 309530), a c.2587C>T change was identified in exon 8 leading to a p.R863W substitution. In the MRX 18 family, a c.2402A>C change in exon 6 was detected, leading to a p.Q801P substitution. A third, unpublished family from the USA (US166) had a c.2273G>A change in exon 5 leading to a p.R758Q substitution. In a fourth, Australian family (AU128) a c.1075C>T change in exon 4 was noted, leading to a p.R359C substitution in the IQ-like domain of IQSEC2. (B and C) Sequence alignments of IQSEC1, IQSEC2 and IQSEC3 showing the location of mutations (bold red type above alignments) in the IQ-like and Sec7 domains. Note that IQ-like motifs lack the G and second basic residue found in canonical IQ motifs. Characters within parentheses can substitute for each other. The R359C mutation disrupts the basic (R) residue. Note that IQSEC2 Sec7 domain mutations do not affect predicted GTP binding residues (bold blue type below alignments) in the structure of the related Sec7 domain in the GEF ARNO. Grey shading indicates amino acids that are identical in all four sequences.

Figure 3 Missense mutations in IQSEC2 that cause non-syndromic XLID reduce guanine nucleotide exchange activity. (A) Radiometric analysis of the effects of mutations in the Sec7 domain of IQSEC2 on guanine nucleotide exchange activity. Recombinant wild-type (dark blue) or mutated versions of the Sec7 domain of IQSEC2 were incubated with recombinant Arf6 and GTPγ³⁵S to catalyze binding of GTPγ³⁵S to Arf6. Arf6-GTPγ³⁵S was isolated on nitrocellulose membrane and the amount of bound radiolabel was quantified in a scintillation counter. Each XLID mutation in the Sec7 domain (light blue) significantly reduced GTP binding to Arf6. *p < 0.0001 vs. wild-type. Note that mutations to E849 (sky blue) are artificial dominant-negative mutants that strongly reduce GEF activity of the Sec7 domain. (B) Guanine nucleotide exchange activity in vivo analyzed using a pull-down assay. Arf6 and either wild-type (dark blue) or mutated (light blue) full-length IQSEC2 were co-expressed in HEK293 cells. The cells were lysed and Arf6-GTP was isolated on beads coated with the adaptor protein Golgi-localized, γ ear-containing Arf binding protein 3 (GGA), which binds to GTP-bound but not GDP-bound Arfs. Arf6-GTP was stripped from the beads and quantified by western blotting. Each mutation reduced activation of Arf6 in this cellular assay.

Table 1 IQSEC family of proteins

Table 2 Guanine nucleotide exchange factors implicated in X-linked intellectual disability

Extra View to: Shoubridge C, Tarpey PS, Abidi F, Ramsden SL, Rujirabanjerd S, Murphy JA, et al. Mutations in the guanine nucleotide exchange factor gene IQSEC2 cause nonsyndromic intellectual disability. Nat Genet 2010; 42:486 - 488; PMID: 20473311; http://dx.doi.org/10.1038/ng.588

 Google Scholar