Unc119 Lipid-Binding Chaperone

Cloning and Expression

Using a subtractive hybridization strategy, Higashide et al. (1996) identified a retina-specific cDNA that they designated HRG4 (human retinal gene-4). Northern blot analysis revealed that the approximately 1.4-kb HRG4 mRNA is expressed specifically in human retina. The authors also cloned a cDNA encoding RRG4, the rat HRG4 homolog. The predicted 240-amino acid human and rat proteins both contain an N-terminal region rich in proline and glycine followed by a region with a mixture of alpha helices, beta sheets, and turns. Sequence comparisons indicated that the proline-glycine domains of RRG4 and HRG4 share only 67% homology, while the rest of the sequence is 100% identical. By in situ hybridization, Higashide et al. (1996) demonstrated that the HRG4 gene is expressed specifically in photoreceptors, both rods and cones, in human retina. In rat, the authors observed high levels of RRG4 expression in the outer retina beginning around postnatal day 5, when the photoreceptors begin to differentiate, and expression increased rapidly to reach the adult level by postnatal day 23.

Swanson et al. (1998) found evidence of alternative splicing of human and bovine UNC119 mRNA, but not of mouse Unc119 mRNA. Northern blot and RT-PCR analyses revealed that the UNC119 gene is expressed at low levels in nonretinal tissues.

Using a yeast 2-hybrid screen to identify proteins that interact with IL5RA (147851), Cen et al. (2003) cloned UNC119 from a fetal liver cDNA library. Western blot analysis showed expression of a 37-kD UNC119 protein in eosinophils and mononuclear cells.

By Western blot analysis, Gorska et al. (2004) showed that UNC119 was expressed in T cells.

Rainy et al. (2016) identified 4 UNC119 paralogs in the zebrafish genome. Based on sequence similarity, unc119a and unc119b are orthologs of mammalian UNC119 and UNC119B, respectively. Zebrafish unc119c is distinct and is not present in mammals. In situ hybridization revealed ubiquitous expression of unc119a and unc119b throughout zebrafish central nervous system. In contrast, unc119c was specifically expressed in retina and pineal gland.

Gene Structure

Swanson et al. (1998) reported that the UNC119 gene contains at least 5 exons and spans 8 kb.

Mapping

By analysis of somatic cell hybrids and FISH, Swanson et al. (1998) mapped the UNC119 gene to 17q11.2. Higashide and Inana (1999) confirmed this localization by somatic cell hybrid analysis.

Gene Function

Mutations in the C. elegans unc119 gene lead to defects in locomotion, feeding behavior, and chemosensation. Both Swanson et al. (1998) and Higashide et al. (1998) observed that HRG4 shares strong homology with the C. elegans unc119 protein, leading Swanson et al. (1998) to designate the human protein UNC119. Swanson et al. (1998) stated that a human UNC119 cDNA functionally complemented the C. elegans unc119 mutation. Using immunofluorescence, Higashide et al. (1998) localized HRG4 to the outer plexiform layer of the retina in the synaptic termini of rod and cone photoreceptors. Electron microscopic immunolocalization showed that the protein is present in the cytoplasm and on the presynaptic membranes of the photoreceptor synapses. The authors suggested that HRG4 may play a role in the mechanism of photoreceptor neurotransmitter release through the synaptic vesicle cycle. They noted that the homology of HRG4 and unc119 is consistent with a possible role of HRG4 in the synaptic vesicle cycle, because the broad effects of unc119 on neuronal function are consistent with a defect in neurotransmission.

Cen et al. (2003) confirmed that UNC119 interacts with IL5RA in eosinophils by coimmunoprecipitation experiments. They found that UNC119 also interacts, through its C- and N-terminal motifs, respectively, with the SH2 and SH3 domains of Src family kinases, such as LYN (165120) and HCK (142370), thereby inducing catalytic activity of the kinases. Eosinophils transduced with UNC119 had increased LYN activity and demonstrated prolonged survival in the absence of growth factors such as IL5 (147850). UNC119 inhibition reduced eosinophil survival.

Using coprecipitation and GST pull-down analysis, Gorska et al. (2004) found that UNC119 interacted with T-cell receptor (TCR; see CD3E; 186830), as well with LCK and FYN (137025). Fluorescent microscopy demonstrated cytoplasmic expression with translocation to the CD3/CD4 (186940) complex upon stimulation. LCK and FYN were activated by either exogenous UNC119 or by overexpression of UNC119. Activation required the presence of the UNC119 SH3-binding motif. Treatment of T cells with UNC119 antisense constructs blocked LCK and FYN activation and, like LCK antisense, blocked TCR signaling pathways. Gorska et al. (2004) concluded that UNC119 is a receptor-associated activator of Src-type kinases.

Using yeast 2-hybrid analysis of a retina cDNA library, Kobayashi et al. (2003) identified ARL2 (601175) as a tissue-specific interactor for HRG4. Western blot analysis and immunofluorescence microscopy showed colocalization of Arl2 and Hrg4 in rat retina.

Veltel et al. (2008) found that recombinant human GTPase-activating protein RP2 (300757) formed a complex with HRG4 and murine Arl3 (604695), a small GTPase. RP2-induced hydrolysis of Arl3-GTP in the Arl3-HRG4 complex led to the release of HRG4, which bound only weakly to Arl3-GDP.

Using immunofluorescence and Western blot analyses of mouse and rat retina cross-sections, Sinha et al. (2013) found that Unc119 localized predominantly to rod inner segments under all levels of illumination. In contrast, with illumination, the majority of rod transducin alpha subunit (GNAT1; 139330) translocated from the rod outer segment to other rod cellular compartments, where it colocalized with Unc119. Western blot analysis showed that mouse retina contained an approximately 1 to 4 molar ratio of Unc119 to Gnat1. Analysis of Gnat1 -/- mouse retina revealed that expression of the Unc119 and Gnat1 proteins was codependent, suggesting that Gnat1 interacts with Unc119 to stabilize it via complex formation.

Biochemical Features

Cheguru et al. (2015) examined the solution structure of the complex formed by myristoylated chimeric GNAT1 and amino acids 50 to 240 of UNC119. They found that upon binding of GNAT1 to UNC119, the N-terminal alpha helix of GNAT1 rotated 45 degrees at the hinge residues 27 to 29 and bent around residues 8 to 9. The analysis also suggested the involvement of a novel interaction interface between the 2 proteins. The effector binding site of GNAT1 was occluded in the complex with UNC119.

Molecular Genetics

Kobayashi et al. (2000) reported that a heterozygous premature termination codon mutation (604011.0001) in the UNC119 gene resulted in late-onset cone-rod dystrophy. In transgenic mice carrying the same mutation, they found age-dependent fundus lesions with accompanying electroretinographic (ERG) changes consistent with defects in photoreceptor synaptic transmission (depressed b-wave, normal c-wave) and retinal degeneration with marked synaptic and possible transsynaptic degeneration.

In a 32-year-old woman, 1 of 3 patients with CD4 lymphopenia in whom known causes had been excluded (IMD13; 615518), Gorska and Alam (2012) identified a missense mutation (G22V; 604011.0002). Noting that a mutation in a different region of UNC119 had been reported in a patient with onset of retinal dystrophy at 40 years of age (604011.0001; Kobayashi et al., 2000), Gorska and Alam (2012) stated that while the immunodeficient patient had no current vision problems, the possibility of future retinopathy could not be excluded.

Animal Model

Kubota et al. (2002) found that expression of a mutant HRG4 protein in the photoreceptor synapses of their transgenic mouse model had intrasynaptic and transsynaptic effects. They noted a decrease in 3 synaptic vesicle proteins, an increase in 5 cytoplasmic and plasma membrane proteins, and a significant reduction in thickness in the inner plexiform layer. These data supported a close relationship of HRG4 with other participants in synaptic vesicle function but noted that the interaction was not mediated by a direct coupling of HRG4 with any of the tested synaptic proteins, but possibly through interaction with a 23-kD protein.

Rainy et al. (2016) found that zebrafish unc119c interacted with arl3l2, which also localized to pineal gland and retina. Knockdown of unc119c and arl3l2 in zebrafish larvae resulted in significantly impaired vision compared with controls. Immunohistologic analysis showed that knockdown of unc119c led to mistrafficking of cone transducin (see 139340), resulting in photoreceptor degeneration.