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. 2016 Sep 21;91(6):1292-1304.
doi: 10.1016/j.neuron.2016.08.022.

Structural Basis for Regulation of GPR56/ADGRG1 by Its Alternatively Spliced Extracellular Domains

Free PMC article

Structural Basis for Regulation of GPR56/ADGRG1 by Its Alternatively Spliced Extracellular Domains

Gabriel S Salzman et al. Neuron. .
Free PMC article


Adhesion G protein-coupled receptors (aGPCRs) play critical roles in diverse neurobiological processes including brain development, synaptogenesis, and myelination. aGPCRs have large alternatively spliced extracellular regions (ECRs) that likely mediate intercellular signaling; however, the precise roles of ECRs remain unclear. The aGPCR GPR56/ADGRG1 regulates both oligodendrocyte and cortical development. Accordingly, human GPR56 mutations cause myelination defects and brain malformations. Here, we determined the crystal structure of the GPR56 ECR, the first structure of any complete aGPCR ECR, in complex with an inverse-agonist monobody, revealing a GPCR-Autoproteolysis-Inducing domain and a previously unidentified domain that we term Pentraxin/Laminin/neurexin/sex-hormone-binding-globulin-Like (PLL). Strikingly, PLL domain deletion caused increased signaling and characterizes a GPR56 splice variant. Finally, we show that an evolutionarily conserved residue in the PLL domain is critical for oligodendrocyte development in vivo. Thus, our results suggest that the GPR56 ECR has unique and multifaceted regulatory functions, providing novel insights into aGPCR roles in neurobiology.

Keywords: X-ray crystallography; adhesion GPCR; monobody; oligodendrocyte development; protein engineering.


Figure 1
Figure 1. Crystal structure of GPR56 extracellular region in complex with high-affinity and specific monobody
(A) Schematic of predicted GPR56 domain structure including ECR composed of unidentified N-terminal domain (cyan), linker (pink), and cleaved GAIN domain (NTF, gray; Stachel, green; autoproteolysis site, *). Though the terms ‘Extracellular Domain’ or ‘Ectodomain’ (both abbreviated ECD) are conventional, we have chosen to refer to the extracellular part of GPR56 as ‘extracellular region’ (ECR) to avoid confusion given that the ECR is composed to two protein domains (Rossmann and Argos, 1981). \\ represents unclear domain/linker boundary. (B) Binding titration of purified mouse GPR56 ECR to yeast-displayed monobody α5. Bound GPR56 was quantified using flow cytometry. (C) Binding signal of purified α5 (25 nM) to HEK293T cells overexpressing full-length mouse GPR56 (+) and control cells (−) detected by flow cytometry. (D) Binding signals of different purified aGPCR extracellular fragments at 250 nM (BAI3, ADGRB3; Lphn1, ADGRL1; Lphn3, ADRGL3; GPR112, ADGRG4; m, mouse; h, human; r, rat) to yeast-displayed α5. (E) Binding signal of α5 and α5_m5 to purified GPR56 ECR-coated M280 beads (see Figure S2B). (F) The crystal structure of GPR56 ECR in complex with α5 (orange). Cys residues involved in a disulfide bond are colored yellow, with the interdomain disulfide bond (C121-C177) indicated by the arrow. The linker and Stachel are colored pink and green, respectively and the asterisk indicates the autoproteolysis site. (G) Close-up view of the binding interface between the PLL domain, PLL-GAIN linker, and GAIN domain. Residues at the binding interface are shown as sticks. The PLL domain, PLL-GAIN linker, and GAIN domain are colored cyan, pink, and gray, respectively. α5 is shown as a transparent orange surface. Polar contacts are indicated by yellow dashes. (H) Crystal structures of autoproteolyzed GAIN domains of GPR56 (top) and Lphn1 (PDB: 4DLQ; bottom) in identical orientations. The α-helices in subdomain A (yellow background) are labeled, and the boxed labels indicate α-helices present in Lphn1 but not GPR56. (I) Human disease-causing GPR56 mutations (red) mapped to the GAIN domain. See also Figures S1–S2.
Figure 2
Figure 2. The PLL domain of GPR56 is a previously unidentified domain that likely diverged from the pentraxin and LNS folds
(A) The PLL domain of GPR56, the PTX domain of C-reactive protein (PDB ID: 3PVN), and the LNS domain of Neurexin-1 beta (PDB ID: 3QCW) in similar orientation. β-Strands are numbered from N to C terminus, and equivalent β-strands are colored in the same manner. Cys residues involved in a disulfide bond are colored yellow. (B) Schematic of β-strand connectivity comprising the two β-sheets of each domain. Wavy arrows represent loops with geometry similar to a β-strand. (C) Human disease mutations (red) mapped to the PLL domain.
Figure 3
Figure 3. Precise deletion of the PLL domain, as in GPR56 splice variant 4, leads to increased basal activity
(A) Domain architecture schematics and function-metrics of important GPR56 constructs (data compiled from D–F, Figure 5 and Table S1). Residue numbers for domain boundaries based on the crystal structure and the interdomain disulfide bond are shown. Function metrics used are: (0) none, (1) very little, (2) less than WT, (3) comparable to WT, (4) more than WT, (ND) not determined. #see also Figure S3. (B) Expected transcripts in two knockout mouse alleles. The starting ATG for WT GPR56 is in exon 2. The S4 variant has its starting ATG in exon 4. The targeting strategy for GPR56(old)−/− mice was to delete exons 2 and 3, which preserved the S4 variant, whereas the GPR56(new)−/− allele deletes exon 4–6, causing a frameshift that leads to a deletion of all splicing variants of GPR56. (C) RT-PCR showing the presence of the S4 transcript in GPR56(old)−/− but absence in GPR56(new)−/− mouse brains. (D–E) In order to quantify cell-surface expression of GPR56 mutant constructs with decreased affinity for α5, IP-western blot was performed. (D) Western blot of whole cell lysates of cells expressing different GPR56 constructs. (E) Western blot of lysate (L) and lysate subject to streptavidin pull-down (P) of HEK293T cells transfected with WT and mutant GPR56 constructs. (F) Basal activity of mutant GPR56 constructs as measured by the SRE-luciferase reporter assay. Top: Basal activity of mutant constructs. Bottom: Basal activity of mutant constructs normalized for cell-surface expression using band densities from E. Data are presented as mean ± S.E.M.; n = 3. sp, signal peptide. See also Figures S3–S4.
Figure 4
Figure 4. The two β-sheets of the PLL domain experienced different evolutionary pressures
(A) Sequence alignment of a segment of the PLL domain from 12 species of GPR56. Conservation scores greater than 3 are shown at the top, with 9 representing the highest conservation. Residues found mutated in BFPP patients (Y88 and C91) are in red. C91, participating in the intra-PLL domain disulfide bond, is highlighted in yellow. H89 is highlighted in maroon. (B) Sequence identities for different fragments of GPR56 between human and the indicated species. *Taken from Makalowski and Boguski (1998). (C) Conservation score of each residue is mapped on the GPR56 ECR structure. Stachel and N-linked glycans are shown as green surface and yellow sticks, respectively. Note that the conserved patch on β-sheet B (solid box, panel D) and the non-conserved patch on β-sheet A (dashed boxes in C and E) are the most and least conserved patches in the entire ECR, respectively. (D) Close-up of the most conserved surface-exposed patch. The sidechains of the residues with the highest conservation score shown as sticks. (E) Pairwise surface conservation between human and mouse GPR56. See also Figure S5.
Figure 5
Figure 5. The conserved patch of the PLL domain is required for GPR56 function in vivo
(A) Injection of WT and mutant mouse GPR56 mRNAs generate embryos with varied levels of CNS myelin basic protein (mbp) expression (black arrow, hindbrain) at 65 hours post-fertilization (hpf). Embryos were given the following scores to signify (0) none, (1) weak, (2) modest, (3) WT, and (4) excess CNS mbp expression. (B) Average CNS mbp score (± S.E.M.) for phenol red (+ Control), WT, H89A, H381S, S150A, and C121S+C177S (C1+C2) injected embryos (from left to right). Injection of WT GPR56 causes an increase in CNS mbp score compared to control-injected embryos (p<7.56×10−6). H381S and H89A abolish the effect of GPR56 overexpression on CNS mbp expression (no significant difference from control injected, significantly less than WT injected: H381S, p<.02; H89A, p<2.68×10−5). S150A and C1+C2 do not affect GPR56-induced CNS mbp overexpression (v. control injected: S150A, p<3.78×10−5; C1+C2, p<.005). (C) Cell surface expression and basal activity of mutant GPR56 constructs as measured by the SRE-luciferase reporter assay. Top: Cell-surface expression of untagged WT and mutant GPR56 constructs with affinity for α5 comparable to WT measured by flow cytometry. Middle: Basal activity of mutant constructs. Bottom: Basal activity of mutant constructs normalized for cell-surface expression using MFI from Top. Data are presented as mean ± S.E.M.; n = 3.
Figure 6
Figure 6. Monobody α5 is an allosteric inverse-agonist for GPR56
(A) Effect of 1 µΜ monobody on GPR56 activation as measured by SRE-luciferase assay in HEK293T cells. Data are presented as mean ± S.E.M.; n = 3; *, p<0.01 compared to Mock by two-tailed Student’s t-test; NS, not significant compared to Mock. (B) SRE-luciferase activity in HEK293T cells is plotted as a function of α5 concentration. Line represents the best fit of the 1:1 binding model for calculation of IC50. (C) Stereo image of the interface between α5 and GPR56 ECR. ECR is colored by conservation score, as in Figure 4C. α5 residues important for GPR56 binding (Figure S2D) shown as sticks.
Figure 7
Figure 7. Working model of mechanisms underlying GPR56 function
(A) Schematic of GPR56 domain structure comparing WT and S4. In all panels, the PLL domain is colored cyan and maroon, corresponding to β-sheets A and B, respectively. (B) Scale model of full-length GPR56 based on the crystal structure of the ECR and a model of the 7TM (generated based on GCGR structure, PDB ID: 46LR). An arbitrary orientation of the ECR with respect to the 7TM is chosen. Residues mutated in BFPP are shown as yellow spheres. H89 is shown as blue spheres. (C) A working model of aGPCR signaling involves ligand-induced activation. In this model, full-length GPR56 is activated when a natural ligand binds to the conserved patch on the PLL domain including H89, causing conformational changes, perhaps including shedding. Introducing the H89A mutation (blue star) to the conserved patch of the PLL domain or deleting the PLL domain completely (as in S4) would result in abrogation of ligand binding and therefore no ligand-induced activation. Binding of α5 likely stabilizes the ECR, causing decreased signaling. sp, signal peptide. See Figure S6 for further possibilities.

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