Adhesion GPCR GPR56 Expression Profiling in Human Tissues

Abstract

Despite the immense functional relevance of GPR56 (gene ADGRG1) in highly diverse (patho)physiological processes such as tumorigenesis, immune regulation, and brain development, little is known about its exact tissue localization. Here, we validated antibodies for GPR56-specific binding using cells with tagged GPR56 or eliminated ADGRG1 in immunotechniques. Using the most suitable antibody, we then established the human GPR56 tissue expression profile. Overall, ADGRG1 RNA-sequencing data of human tissues and GPR56 protein expression correlate very well. In the adult brain especially, microglia are GPR56-positive. Outside the central nervous system, GPR56 is frequently expressed in cuboidal or highly prismatic secreting epithelia. High ADGRG1 mRNA, present in the thyroid, kidney, and placenta is related to elevated GPR56 in thyrocytes, kidney tubules, and the syncytiotrophoblast, respectively. GPR56 often appears in association with secreted proteins such as pepsinogen A in gastric chief cells and insulin in islet β-cells. In summary, GPR56 shows a broad, not cell-type restricted expression in humans.

Keywords: ADGRG1; GPR56; microglia; thyroid.

Figure 1

Verification of GPR56 Abs using tagged GPR56. (a) Schema of GPR56 and GPR56 Ab binding sites; see running text for explanations. (b–e) COS-7 cells, transfected with C-terminal myc-tagged or untagged full-length (FL) ADGRG1 (NM_201524.3), or ΔNTF ADGRG1 pcDNA3.1 were used to examine GPR56 Ab specificity. (b) Monolayered, acetone-fixed GPR56myc cells were costained for GPR56 and myc. All GPR56 Abs bound GPR56 specifically (arrows: membrane-near staining); immunofluorescence. The GPR56^CTF Ab binds only the untagged GPR56 C-terminus, therefore, cells transfected with untagged ADGRG1 were co-stained with the well-established GPR56^NTF1 Ab. (c) GPR56myc cells were detached and paraffin-embedded. Sections were costained with the indicated Abs (see also (b)); immunofluorescence. The GPR56^NTF1 Ab unspecifically stained myc-negative cells (arrows). (d) Flow cytometry: GPR56myc Cos-7 cells were analyzed for GPR56 cell surface expression; MFI: mean fluorescence intensity. (e) Western blotting: lysates of mock, full-length (FL) GPR56myc or ΔNTF GPR56myc cells were blotted and the indicated Abs applied. The GPR56^NTF4 Ab bound to uncleaved GPR56 (~86.5 kDa), the cleaved NTF (56.6–60.5 kDa multiple bands), and, inconstantly and weakly, the CTF (~26 kDa). The myc Ab confirmed the results; * likely unspecific bands.

Figure 2

Verification GPR56 Abs comparing BEN cells with ADGRG1-deficient BEN clones. (a) Human cancer cell lines were analyzed for GPR56 cell surface expression with the GPR56^NTF1 Ab in flow cytometry. BEN cells, strongly GPR56-positive, were used for generating GPR56 knock-out (KO) clones; MFI, mean fluorescence intensity. (b) BEN cells were stained with GPR56 Abs before (left) or after (right) paraformaldehyde-fixation in flow cytometry. (c) Selection of BEN cell surface GPR56-negative clones. Left: BEN cells were transfected with ADGRG1 sgRNA CRISPR/Cas9-GFP plasmid. After 24 h, the cells were stained with the GPR56^NTF1 Ab, GPR56-negative GFP-positive cells (red bordered) were FACS-sorted and grown as clones. Right: two clones of each ADGRG1 sgRNA were selected (named KO1/1 etc.), none expressed GPR56 at the cell surface; flow cytometry. (d) Quantitation of ADGRG1 by qRT-PCR. RPL27-normalized log2 x-fold ADGRG1 mRNA levels of BEN GPR56KO clones compared with BEN WT cells, which were set to 1 (mean, n = 3). (e) RNA-sequencing data of BEN WT and two GPR56KO clones. Present ADGRG1 transcripts were named according to the Ensembl database. Left: Abundance of transcripts is given as FPKM (fragments per kilobase per million mapped reads) and visualized by a heat map. Right: Visualization of the present transcript variants. The black boxes represent the transcribed exons. Transcription start sites differ obviously. Binding sites of the ADGRG1 sgRNA1 (used to generate GPR56KO1 clones) and 2 (GPR56KO2 clones) are indicated by arrows. Arrowhead: translation start site. The grey columns indicate regions where GPR56 domains are encoded. The scheme was generated according to data from Uniprot and Ensembl database (http://wormweb.org/exonintron, accessed on 30 October 2020). (f) Monolayered, paraformaldehyde-fixed BEN WT and GPR56KO2/1 cells were stained with the indicated GPR56 Abs; immunofluorescence. GPR56KO2/1 cells are completely GPR56-negative. (g) Detached BEN WT and GPR56KO2/1 cells were each injected into an isolated mouse lung (serving as a scaffold) which was paraffin-embedded afterwards. Sections were stained with the GPR56^NTF4 Ab; immunohistochemistry. (h) Western blotting: lysates of mock- and ADGRG1-transfected COS-7, BEN WT, and GPR56KO cells were blotted and the GPR56^NTF4 Ab applied; arrowhead, this specific band also appeared slightly in GPR56KO1/2 cells; * likely unspecific bands.

Figure 3

ADGRG1 and GPR56 expression correlate well in normal human tissues. (a,b,d–g) Immunostained paraffin sections using the GPR56^NTF4 Ab. (a) In the spleen, GPR56-positive cells, likely cytotoxic T cells, are located in the mantle zone (arrowheads); *, white pulp artery. (b) In pancreatic islets, GPR56 and insulin co-localize in β-cells; double-immunofluorescence. (c) Transcriptomic profile of adhesion GPRCs of the G subfamily (ADGRG1-G7) in normal human tissues, bulk RNA-sequencing data [20], reads per kilobase exon per million mapped reads (RPKM) are given. (d) The liver is GPR56-negative confirming the ADGRG1 data (c). β-catenin was stained as a positive control (cv central vein). (e) The thyroid showed the highest ADGRG1 levels (c). Thyrocytes (arrowheads) are strongly GPR56-positive (coll colloid); staining is attenuated at the apical side. (f) Kidney tubules express GPR56. Left: pt proximal and dt distal tubules; *, vascular pole of the glom glomerulus. Right: Double-immunofluorescence labeling of GPR56 and tubule-specific lectins. Lotus tetragonolobus lectin (LTL) binds to proximal tubules, Dolichos biflorus agglutinin (DBA) to distal tubules. (g) In a full-term placenta (mat bl maternal blood, fv fetal vessel), GPR56 appeared attenuated at the basal side of the syncytiotrophoblast (arrowheads).

Figure 4

GPR56 is expressed in IBA-1-positive microglia. (a–c) Immunostained brain paraffin sections using the GPR56^NTF4 Ab. (a) Left: GPR56-immunoreaction in the temporal cortex (A22) grey (GM) and white matter (WM). Middle: GPR56-positive cells, probably microglia cells, in the GM in all layers. Right: GPR56-positive cells, likely microglial cells, in the WM. (b) Double immunofluorescence labeling of GPR56 and IBA-1 in the temporal cortex GM. Nuclei were stained with DAPI. All IBA-1-positive microglial cells were GPR56-positive. (c) GPR56-positive isolated cells, likely astroglia cells, in the temporal cortex GM.

Figure 5

GPR56 localization changed during thyroid tumorigenesis. (a) In a follicular carcinoma, GPR56 is located mainly intracellularly as in normal thyrocytes. (b,c) In papillary carcinomas, GPR56 was strongly upregulated in lateral tumor cell contacts (inserts, arrowheads). Interestingly, at the invasion front GPR56 vanished ((c), right insert) and tumor cells located in the invaded capsule showed no membranous staining. (d) In a poorly differentiated (left) and in an anaplastic carcinoma (right) GPR56 was strongly downregulated or even disappeared, respectively.