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Bilateral frontoparietal polymicrogyria is an autosomal recessively inherited human brain malformation with abnormal cortical lamination. The affected cortex appears to consist of numerous small gyri, with scalloping of the cortical-white matter junction. There are associated white matter, brainstem and cerebellar changes. Affected individuals present with mental retardation, language impairment, motor developmental delay and seizure disorder. GPR56 is the causative gene. Here we report a novel missense mutation of GPR56, E496K, identified in a consanguineous pedigree with bilateral frontoparietal polymicrogyria. GPR56 is cleaved at the G protein-coupled receptor proteolytic site into an N- and a C-terminal fragment, named GPR56N and GPR56C respectively. E496K is located in GPR56C. Further biochemical studies show that this mutation affects GPR56C cell surface expression similar to the effect of a previously reported mutation, R565W. These results provide further insights into how GPR56 mutation causes neurological disease.
Polymicrogyria is a heterogeneous term used to describe a type of cortical malformation that is characterized by numerous (poly) small (micro) gyri overfolded upon one another . Bilateral frontoparietal polymicrogyria is a recently defined autosomal recessive brain malformation [2, 3]. The affected individuals present with severe neurological impairment. The affected cortex has an irregular surface and appears to comprise of an excessive number of small gyri, resulting in a scalloped appearance of the cortical-white matter junction, a shared feature with polymicrogyria. The observed cortical abnormality extends diffusely across the frontal and parietal lobes with a decreasing anterior-to-posterior gradient of severity. There are associated myelination defects, with areas of abnormal signal in the cerebral white matter, and cerebellar cortical dysplasia, as well as mild hypoplasia of the pons and cerebellar vermis. The radiological features seen in bilateral frontoparietal polymicrogyria show many similarities to those seen in conditions with cobblestone complex, such as muscle-eye-brain disease, Fukuyama’s congenital muscular dystrophy, and Walker-Warburg syndrome . The causative gene of bilateral frontoparietal polymicrogyria is GPR56 [5, 6]. Further histological study in mice with deletion of the Gpr56 gene as well as post-mortem human brain specimen harboring GPR56 mutation confirmed that the histopathology of bilateral frontoparietal polymicrogyria is indeed a cobblestone-like cortical malformation [7, 8].
GPR56 is an orphan G protein-coupled receptor that belongs to the family of adhesion G protein-coupled receptors . GPR56 mRNA is selectively expressed in hematopoietic stem cells and neural progenitors, suggesting a role in multipotent cell identity and tissue development . In the developing mouse brain, GPR56 mRNA is detected in the embryonic ventricular zone, the site for neural progenitor cells [5, 7]. Like other adhesion G protein-coupled receptors family members, GPR56 has a very long N-terminal stalk and seven transmembrane domains. The GPR56 protein undergoes G protein-coupled receptor proteolytic site mediated autoproteolytic process, resulting in an N- and a C-terminal fragment, named GPR56N and GPR56C respectively [11–13]. GPR56N has been found to be associated with GPR56C as well as secreted into the cultured media of GPR56-expressing cells . GPR56C is a plasma membrane-bound fragment . The biological consequence of GPR56 protein cleavage and the functional interaction of GPR56N and GPR56C remain largely unknown.
Here we report a novel GPR56 mutation E496K found in a bilateral frontoparietal polymicrogyria patient. Further biochemical analysis indicates this mutation affects the cell surface expression of GPR56C.
The proband is the eldest of 3 children born to a first-cousin couple of Yemeni origin. The pregnancy, delivery and perinatal period were unremarkable. His two younger sisters are well with no noticeable medical problems. Apart from sickle cell anemia in the father, there are no significant anomalies and neurological disorders in the family.
The proband’s neonatal period was uneventful, but he was found to have delayed developmental milestones at 8 months of age. He subsequently developed tonic-clonic seizures. When assessed at 2-year-and-9-months of age, he was hypotonic and developmentally delayed. He was able to cruise, but could not walk independently. Speech was limited to a few isolated words. Physical examination revealed relatively long palpebral fissures and a broad forehead, although obvious dysmorphic features were absent. Detailed ophthalmological assessment was unremarkable aside from right esotropia. On follow-up examination at the age of 6-year-and-6-months he was still hypotonic and severely developmentally delayed. He was able to walk independently short distances, albeit very unsteadily, and dribbled constantly. His seizure control had improved markedly on commencing sodium valproate therapy, although he continued to have 2–3 seizures daily. His speech was limited to his parents’ names. He mainly communicated by gesturing and crying. He remained dependent on adults for all his needs, requiring help with feeding, dressing and toileting. On physical examination his occipitofrontal head circumference measured on the 9th centile at 51cm, having been on the 75th centile during the first year of life and between 25th to 50th centiles at 2-year-and-9-months. Laboratory results for CK and lactate were within normal ranges and the chromosome karyotype was normal as well. Brain magnetic resonance imaging (MRI) findings are described in detail below.
Sequence analysis was performed with DNA from the proband; subsequently we did targeted mutation analysis in the DNA of both parents. DNA was extracted from peripheral blood leukocytes. Genomic DNA was used as a template for polymerase chain reaction. All coding exons 2–14 and exon/intron boundaries of the GPR56 gene were sequenced (AF106858/NM_005682 transcript variant 1, isoform a), as previously described .
All expression constructs were generated using standard molecular biology cloning methods as previously mentioned . VSVG/His-tagged GPR56 was generated in pCS2+ vector (gift from Dr. Xi He) . Primers used for the E496K mutagenesis include the forwarding primer: 5′-TCCTGGATGGGCCTCAAGGGCTACAATCTCTACC -3′ and the reverse primer: 5′-GGTAGAGAGATTGTAGCCCTTGAGGCCCATCCAGGA-3′. All expression constructs were sequenced to confirm their identity.
The C-terminal peptide of GPR56 (CGGPSPLKSNSDSARLPISSGSTSSSRI) were synthesized, conjugated to keyhole limpet hemocyanin, and injected into rabbits for antiserum production (Zymed Laboratories, San Francisco, CA).
HEK293T cells were maintained in Dulbecco’s Modified Eagle Medium with 10% heat inactivated fetal bovine serum and incubated at 37°C with 5% CO2. Cells were transiently transfected with different expression constructs using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to manufacturer’s protocol.
Western blot and biotinylation of cell surface proteins were performed as described before . HEK293T cells were transfected with VSVG/His tagged wild type or mutant GPR56 constructs.
Multiplanar MR imaging of the brain was performed on the proband at 5 years of age (Figure 1). On initial review, the cortex of the frontal and parietal lobes appeared to be increased in thickness. On closer inspection, the cortex was seen to consist of multiple small gyri, resulting in a scalloped appearance of the cortical-white matter junction. Both cerebral hemispheres were equally and symmetrically affected, with a decreasing anterior-to-posterior gradient of involvement, the frontal lobes being most affected, the parietal lobes somewhat less so and the occipital and temporal lobes relatively spared. There were asymmetric areas of abnormal signal in the white matter of both cerebral hemispheres. Both lateral ventricles were enlarged. There was mild hypoplasia of the inferior cerebellar vermis and pons and subtle areas of disordered folia in the cerebellar hemispheres. These radiological findings are entirely consistent with those described in previous affected individuals with GPR56 mutations [3, 6].
DNA sequence analysis of the proband revealed a homozygous pattern for a G→A mutation at base pair 1486 from the ATG resulting in a Glu→Lys change at the amino acid 496 (Fig 2A). Heterozygosity for the E496K change was found in both parents, confirming homozygosity in the patient (Fig 2A). E496K is located in the 3rd transmembrane spanning region of GPR56 (Fig 2B). Sequence alignment of this region of GPR56 from nine different species, including Homo sapiens, Mus musculus, Rattus norvegicus, Pan troglodytes, Gallus gallus, Bos taurus, Pongo abelii, Macaca mulatta, and Canis lupus familiaris, confirms this is a highly conserved amino acid, suggesting an indispensable role in protein folding and function (Fig 2C).
GPR56 undergoes a G protein-coupled receptor proteolytic site mediated proteolytic process resulting in two fragments, GPR56N and GPR56C [11–13]. The E496K mutation is located in the GPR56C as seen in the two previously reported C-terminal mutations, R565W and L640R. To characterize the biochemical significance of these three C-terminal mutations, we generated N-terminally VSVG-tagged and C-terminally His-tagged GPR56 constructs containing E496K, R565W and L640R point mutations. HEK293T cells were transiently transfected with VSVG/His-tagged wild type GPR56 or three mutants, E496K, R565W, and L640R. Cell lysates were analyzed for cleavage products. Anti-VSVG Western blot analysis of the wild type GPR56 and the three C-terminal mutants revealed GPR56N to be a predominant band around 60 kDa, in addition to a ladder of higher molecular weight protein bands that reflects the heavily N-glycosylated GPR56N (Fig 3A). We detected less GPR56N ladder in cells expressing E496K and R565W mutants.
Anti-GPR56C antibody 199 Western blot analysis detected a band of ~22 kDa in wild type GPR56 expressing cells as well as cells transfected with the three C-terminal mutants, although a decreased level of GPR56C were found in cells expressing E496K and R565W mutants (Fig 3A). These results indicate that these C-terminal mutations did not affect GPS-mediated GPR56 cleavage. The decreased level of GPR56C in GPR56 E496K - and GPR56R565W-expressing cells suggests that these two mutations may affect the stability of GPR56C.
To evaluate the effect of E496K mutation on GPR56 protein cell surface expression, we performed biotinylation experiments. HEK293T cells transfected with wild type or mutant GPR56 were labeled with Sulfo-NHS-Biotin, a membrane impermeable biotinylation agent. Only cell surface-expressed proteins are labeled by Biotin. The cells were lysed and all biotinylated proteins were isolated by streptavidin affinity chromatography. Using Western blot with anti-VSVG or Anti-GPR56C antibody 199, we detected both biotinylated GPR56N and GPR56C in the cells expressing wild type GPR56 and L640R at a comparable level. However, there was only trace amount of biotinylated GPR56C detected in cells expressing E496K and no biotinylated GPR56C in cells expressing R565W (Fig 3B). The level of the 60 kDa predominant band of GPR56N is comparable in wild type GPR56 and its three C-terminal mutants. Interestingly, we detected a much lower amount of the higher molecular weight GPR56N ladder.
Protein cleavage is essential for the function of GPR56, an inability of such cleavage results in bilateral frontoparietal polymicrogyria . However, there are many unanswered questions; what is the biological meaning of this cleavage? Do GPR56N and GPR56C traffic independently? In order to answer these questions, it would be essential to understand how the two GPR56 fragments interact and affect brain development. The documented mutations of GPR56N in patients with bilateral frontoparietal polymicrogyria have been found to interfere with intracellular trafficking as well as higher order of glycosylation of the protein . Although two mutations in GPR56C have been reported previously, functional implications of the C-terminus mutations have yet to be understood.
Our results showed that the three C-terminal mutations did not affect the proteolytic process of GPR56. However, both E496K and R565W mutants showed much less GPR56C that expressed on the cell surface, in comparison to their counterpart GPR56N. This finding indicates the possibilities that GPR56N and GPR56C may traffic independently, as demonstrated in latrophilin, another member of adhesion GPCRs . Further studies of GPR56 protein trafficking and signal transduction are underway.
We have previously shown that GPR56N is heavily N-glycosylated . Proteins undergo primary N-glycosylation in the endoplasm reticulum (ER). Higher order glycosylation modification occurs in the Golgi. The GPR56N protein ladders detected by anti-VSVG Western blot represent the complex carbohydrate chains that are added in the Golgi . We detected significantly less GPR56N ladders in cells expressing E496K and R565W mutants, suggesting the mutations in the GPR56C may somehow affect the intracellular trafficking of GPR56N into Golgi.
We thank Dr. Xi. He, PhD, for providing VSVG-tagged pCS2+ vector. This research was supported in part by NINDS grant R01 NS057536 (X.P.), and a Flight Attendant Medical Research Institute Young Clinical Scientist Award (Z.J).
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