No evidence that human GIGYF2 interacts with GRB10: implications for human disease

Abstract

GIGYF2 (growth factor receptor-bound protein 10 [GRB10]-interacting GYF [glycine-tyrosine-phenylalanine] protein 2) reduces mRNA stability and translation via microRNAs, ribosome quality control, and several RNA-binding proteins. GIGYF2 was first identified in mouse cell lines as an interacting partner with GRB10, which binds to the insulin receptor and the insulin-like growth factor receptor 1. Mutations in the human GIGYF2 gene were reported in autism. In mouse models, Gigyf2 mutations engender several diseases. It was therefore thought that the GIGYF2-associated disease in humans is caused by defective GRB10 signaling. We show here that GIGYF2 does not interact with GRB10 in human cell lines, as determined by co-immunoprecipitation and proximity ligation assays. The lack of interaction is explained by the absence of the critical GYF domain-binding PPGΦ sequence in the human GRB10 protein. These results contrast with the current understanding that a GIGYF2/GRB10 complex is associated with human disease via insulin receptor and insulin-like growth factor receptor 1 signaling and underscore alternative mechanisms responsible for the observed phenotypes associated with mutations in the human GIGYF2 gene.

Figure 1.. Key GIGYF2 mutations linked to autism.

The different motifs of GIGYF2 are indicated: 4EHP stands for eIF4E homologous protein; DDX6 refers to DEAD (Asp-Glu-Ala-Asp)-box helicase 6; GYF stands for glycine–tyrosine–phenylalanine.

Figure S1.. Phylogenetic illustration of the PPGΦ motif in vertebrate orthologs.

Maximum likelihood tree inferred with RAxML using the GTRCAT substitution model from Clustal Omega–aligned orthologous transcript sequences containing the PPGΦ motif. Branch lengths represent nucleotide substitutions per site (scale = 0.1). Species in red encode either the PPGΦ motif (CCTCCAGGCTTT) or a derived variant (CCTCCAGGATTT). The National Center for Biotechnology Information (NCBI) Reference Sequence (RefSeq) accession numbers for corresponding mRNAs are indicated.

Figure 2.. PPGΦ motif of the GRB10 protein is present only in species of the Muridae family of rodents.

Multiple sequence alignment of GRB10 proteins of different species. The alignment was performed using Clustal Omega (Sievers et al, 2011). Amino acids that are similar across all analyzed species are shaded in black. The PPG motifs are highlighted in red. The species names of the respective amino acid sequences are listed on the left. Amino acid sequences of GRB10 from a total of 29 species were analyzed for multiple sequence alignment. The binomial nomenclatures and common names (shown within brackets) are as follows: Mus musculus (House mouse), Mastomys coucha (Southern multimammate mouse), Mus caroli (Ryukyu mouse), Mus pahari (Gairdner’s shrewmouse), Rattus rattus (Black rat), Rattus norvegicus (Brown rat), Grammomys surdaster (African woodland thicket rats), Arvicanthis niloticus (African grass rat), Apodemus sylvaticus (Wood mouse), Jaculus jaculus (Lesser Egyptian jerboa), Marmota marmota (Alpine marmot), Nannospalax galili (Middle East blind mole-rat), Heterocephalus glaber (Naked mole-rat), Homo sapiens (Human), Pan troglodytes (Chimpanzee), Hylobates moloch (Silvery gibbon), Macaca mulatta (Indochinese rhesus macaque), Pongo pygmaeus (Bornean orangutan), Symphalangus syndactylus (Siamang), Gorilla gorilla (Western gorilla), Pan paniscus (Bonobo), Bos taurus (Cattle), Sus scrofa (Wild boar/swine), Canis lupus familiaris (Dog), Panthera tigris (Tiger), Panthera leo (Lion), Panthera onca (Jaguar), Danio rerio (Zebrafish), and Xenopus laevis (African clawed frog).

Figure 3.. PPGΦ motif of the human GRB10 is not present because of an alternative splicing event.

(A) Schematic representation of the structure of murine and human genes encoding GRB10. Gray shading indicates coding sequence (CDS); thick lines and exon numbering indicate the splicing pattern of the major isoform according to Matched Annotation from NCBI and EBI (MANE), whereas thin lines indicate alternative splicing. White exonic parts indicate untranslated regions (UTRs). Light gray–shaded areas indicate orthologous regions of exons. (B) Comparison of the PPGΦ motif encoding sequences in human (Hsa) and mouse (Mmu) genes encoding GRB10. The splice sites are indicated in blue, and the alternative exon in yellow. (C) Schematic model illustrates the design of a set of primers amplifying sequences of human and mouse GRB10 genes flanking the exon encoding the PPGΦ motif. The exons and introns are represented by rectangles and lines, respectively. The exon encoding the PPGΦ motif is shown as a “red” rectangle, and flanking exons are colored in gray. Arrowheads depict the location of the forward and reverse primers used to detect the exon encoding PPGΦ motif in panel “(D)”. (D) RT–PCR analysis of total RNAs derived from mouse cortex and CHX-treated human HAP1 and RPE1 cells using primers described in “(C)”. The PCR products were resolved on a 2.5% agarose gel. The size of the skipped exon is 265 base pairs in humans and 277 base pairs in mice. The arrowhead indicates the skipped exon (minor band) in mice. The asterisk represents a band of unknown identity that is not the expected size for the target splice variant, which is only amplified in the samples of mouse origin.

Figure S2.. Detection of NMD isoforms in CHX-treated cells.

RT–PCR for SRSF2 and SRSF6 was performed as a positive control for the CHX assay, which inhibits the NMD pathway and allows for the detection of unstable or transiently expressed transcripts. The sizes of the bands corresponding to NMD isoforms are indicated in bold.

Figure 4.. Analysis of interactions between human GIGYF2 and GRB10 proteins.

(A) co-IP analysis of endogenous GIGYF2 protein interactions in HEK293T cells. Immunoprecipitation was performed using an anti-GIGYF2 antibody, followed by Western blot analysis with the indicated antibodies. (B) co-IP analysis of endogenous GRB10 protein interactions in HEK293T cells. Immunoprecipitation was performed using an anti-GRB10 antibody, followed by Western blot analysis with the indicated antibodies. (C) PLA for detecting interactions between FLAG-GRB10 and v5-IGF-1R or v5-GIGYF2. PLA signals are shown in yellow; the nucleus and actin cytoskeleton were counterstained with DAPI and phalloidin, respectively. Scale bar = 10 μm. The bar graph represents the average number of PLA signals from at least 30 cells per sample (n = 3 independent experiments). (D) Western blot analysis of cell lysates from PLA shown in panel “(C)”. (E) Analysis of endogenous GIGYF2- and GRB10-containing complexes by size-exclusion chromatography. A total of 10 mg of protein from HEK293T cells was loaded onto a Superose 6 column and run at a flow rate of 0.4 ml/min. Fractions of 0.5 ml were collected, and 50 μl of each fraction was analyzed by Western blotting.

Figure S3.. Molecular interaction network of the human GIGYF2 protein predicted by the STRING database (https://string-db.org/).