Functions and mechanisms of the GPCR adaptor protein Norbin

Abstract

Norbin (Neurochondrin, NCDN) is a highly conserved 79 kDa adaptor protein that was first identified more than a quarter of a century ago as a gene up-regulated in rat hippocampus upon induction of long-term potentiation. Most research has focussed on the role of Norbin in the nervous system, where the protein is highly expressed. Norbin regulates neuronal morphology and synaptic plasticity, and is essential for normal brain development and homeostasis. Dysregulation of Norbin is linked to a variety of neurological conditions. Recently, Norbin was shown to be expressed in myeloid cells as well as neurons. Myeloid-cell specific deletion revealed an important role of Norbin as a suppressor of neutrophil-derived innate immunity. Norbin limits the ability of neutrophils to clear bacterial infections by curbing the responsiveness of these cells to inflammatory and infectious stimuli. Mechanistically, Norbin regulates cell responses through binding to its interactors, in particular to a wide range of G protein-coupled receptors (GPCRs). Norbin association with GPCRs controls GPCR trafficking and signalling. Other important Norbin interactors are the Rac guanine-nucleotide exchange factor P-Rex1 and protein kinase A. Downstream signalling pathways regulated by Norbin include ERK, Ca2+ and the small GTPase Rac. Here, we review the current understanding of Norbin structure, expression and its roles in health and disease. We also explore Norbin signalling through its interactors, with a particular focus on GPCR trafficking and signalling. Finally, we discuss avenues that could be pursued in the future to increase our understanding of Norbin biology.

Keywords: GPCR; NCDN; Norbin; P-Rex1; Rac; neurochondrin.

Figure 1.. Norbin orthologs in animals.

(A) Norbin gene structure highlighting its 7 exons (red boxes) and introns (red lines). In humans, NCDN is located on chromosome 4: 126 637 543–126 647 202. (B) Norbin (NCDN) gene tree in animals (left) generated by the Ensembl gene orthology/paralogy prediction pipeline using the Mus musculus Ncdn gene (ENSMUSG00000028833) as the reference sequence. Norbin is highly conserved throughout vertebrates, and is also found in invertebrates such as Drosophila melanogaster. The homology with the sea squirt Ciona intestinalis is limited, there is only a short fragment of similarity in the C-terminus, and the gene seems to have been lost from worms, as there is no Norbin in Caenorhabditis elegans. In mammals, birds and most fish, a direct 1-to-1 ortholog of human Norbin exists. In some fish species, gene duplication events have occurred, such as in carp, where there are two full-length copies with ∼85% identity. Highly conserved blocks of genomic sequences are indicated with green boxes (right).

Figure 2.. Norbin orthologs in plants.

Norbin (ncdn) gene tree in plants (left), generated by the Ensembl gene orthology/paralogy prediction pipeline, using the Arabidopsis thaliana ncdn gene (AT4G32050) as the reference sequence. In plants, the protein is smaller than in animals (619 amino acids in A. thaliana compared with 729 in humans), but it is highly conserved between species. Highly conserved blocks of genomic sequences are indicated with green boxes (right). Human and D. melanogaster Norbin are included for reference.

Figure 3.. Predicted Norbin structure.

Structural predictions for (A) human Norbin (UniProt: Q9UBB6) and (B) A. thaliana Norbin (UniProt: Q5E911) were obtained from the AlphaFold Protein Structure Database (alphafold.ebi.ac.uk). One highly conserved region between the two species is indicated with red arrows for reference. AlphaFold produces a per-residue confidence score (pLDDT) between 0 and 100. Regions below 50 pLDDT in isolation are considered unstructured.

Figure 4.. Distribution of protein-coding Norbin transcripts in brain regions of newborn mice.

Bam files from the following ENCODE RNAseq datasets were analysed for the abundance of confirmed and putative protein-coding Norbin transcripts in various brain regions of newborn wild-type mice: ENCFF701BYJ, cerebral cortex. ENCFF760YQJ, forebrain. ENCFF983VMB, hindbrain, ENCFF165PCY, hippocampus. ENCFF398QVK, midbrain. Animals were aged between postnatal days 0 and 10, and were of both sexes. To analyse splice variants, intronic reads were quantitated in Seqmonk through read position probe generation over the Ncdn gene followed by exact overlap quantification, normalised to the total read count, with filtering to remove probes with quantitation <100. IDs of the known transcripts are according to Ensembl (v102). Transcript-associated protein forms are defined from Ensembl (where known) or as the amino acid sequence of the longest open reading frame generated by the transcript. The proteins that would result from these transcripts are full-length Norbin (FL, aa 1–729, blue shades), the known 712 aa splice variant (SV, aa 18–729, green shades), and a hypothetical 579 aa splice variant starting in exon 3 for which there is currently no experimental evidence (O, aa 151–729, black).

Figure 5.. Norbin binding of phosphatidic acid.

(A) Total lysate (TL) of COS-7 cells expressing full-length myc-tagged Norbin was subjected to pull-down with immobilised phosphatidic acid (PA) or control phosphatidylinositol (4,5)-diphosphate (PIP₂) as described in [27] and was western blotted with myc antibody. One percent of the TL was loaded for comparison. (B and C) Myc-tagged full-length Norbin and the indicated mutants were subjected to PA pull down as in (A). (D) GFP-tagged N-terminal or internal fragments of Norbin were expressed in COS-7 cells and the TL subjected to PA pull down as in (A) and were western blotted with GFP antibody. (E) GST-tagged N-terminal or internal fragments of Norbin were expressed in E. coli and the TL was subjected to pull down with PA or PIP₂ beads as in (A) and was western blotted with GST antibody. (A,C–E) Western blots shown are representative of three independent experiments.

Figure 6.. Map of Norbin residues with known function.

Binding regions of mGlu₅ (blue box) [34], MCHR1 (yellow box) [25], PKA regulatory subunit RIIα (orange boxes) [5] and Semaphorin 4C (pink box) [52] are mapped. The N-terminal 100 amino acids of Norbin, which stimulate neurite outgrowth [11], are highlighted (green box). Palmitoylation of cysteines C3 and C4 is critical for endosomal localisation of Norbin in neuronal dendrites [10]. The alternative start codon for the splice variant (isoform-2, M18) [4] is highlighted above, as are known point mutations, including five of clinical relevance (bold). E433Q, R478E, W652R and P652L are found in patients with developmental delay, intellectual disability and epilepsy [50]. All four mutations impair Norbin-dependent neurite outgrowth. Additionally, W652R and P652L mutations in/near the mGlu₅-binding region impair mGlu₅ signalling, whereas E433Q and R478E do not [50]. P402* is found in a patient with frontotemporal dementia, and causes altered morphology of FUS-positive granules [54]. A687G is a mutation that was serendipitously found to block Norbin interaction with mGlu₅ [14].