A distinctive activation mechanism of a Family B orphan GPR56 by relocating its shed N-terminal domain

A study on GPR56, belonging to a subfamily of an adhesion GPCR (a.k.a. LN-7TM) of Family B, found that this receptor can be activated by removing its large N-terminal domain. The N-terminal domain (NT) is proteolytically cleaved in a process termed receptor shedding. The cleaved NT associates with the receptor as it maintains itself at an inactive state.
In their suggested model of GPR56 activation, the receptor generates signals upon NT dissociation by means of either: heteromeric interaction between NT and its ligand unidentified; and/or homophilic associations between local NTs.

The GPR56 N-Terminus Controls Receptor Signaling Activity 2011. JBC in press.
KJ Paavola, JR Stephenson, SL Ritter, SP Alter & RA Hall
Department of Pharmacology, Emory University School of Medicine, Atlanta, GA, USA

The study demonstrated:
1. The shed NT of GPR56 (GPR56-NT) associates with the receptor.

2. The NT truncation of GPR56 enhances receptor induction of Rho signalling. The truncated receptors (ΔN-342) showed notably enhanced β-arrestin binding, and were subjected to extensive ubiquitination. The increased cytotoxicity (measured by LDH levels) by the truncated was observed but was reversed by an increase in β-arrestin2.

3. GPR56-NT can homomerise.

Relevance to other studies:
The findings of this study provides an explanatory evidence for an observation made in other study: anti-GPR56 polyclonal antibodies raised against the N-terminal domain of GPR56 activated Rho pathway (Iguchi et al. 2008). Homodimerisation of N-terminal domains have also been documented for other member of the LN-7TM subfamily: Cadherin EGF LAG seven-pass G-type receptor (Celsr) type 2 or type 3 involved in neurite growth (Shima et al. 2007); the N-terminal domain of Celsr2/3 are enormous, over 2000 and over 3000 amino acid lengths respectively.

About GPR56:

Human GPR56 on uniprot
Its N-terminal region comprises 377 amino acids following a signalling peptide of 25 amino acid lengths. Numerous Ser/Thr residues (≈16% of the 377 residues) could be glycosylated in the Golgi apparatus. The N-terminal region contains a GPCR proteolytic site (GPS) located by TM1.

Mutations in GPR56 and the Roles of GPR56 in CNS
Missense mutations in GPS (C346S and W349S) could impair the proteolytic cleavage, and the mutant receptors were found not to traffic beyond the endoplasmic reticulum (Jin et al. 2007).

Loss-of-function mutations in GPR65 result in bilateral frontoparietal polymicrogyria (BFPP) (Piao et al. 2004), an autosomal recessive disorder affecting brain development (see a note below). All identified BFPP-related mutations are in the extracellular domains of the receptor, affecting its cell-surface expression and/or receptor shedding (Jin et al. 2007; Chlang et al. 2011).

Bilateral frontoparietal polymicrogyria (BFPP)
A congenital brain malformation displaying abnormal groove patterning on the surface of the cortex with numerous but smaller gyri. It causes developmental delay in both mental and motor skills, and individuals affected may suffer from epilepsy.
For more information, see a review by Jansen & Andermann 2005.

GPR56 also regulates granule cell adhesion during rostral cerebellar development (Koirala et al. 2009).

GPR56 Signalling and Its Regulation
During the development of the forebrain, it regulates neural progenitor cell migration through activation of G12/13 and Rho pathway involving serum-responsive element and NFκβ-responsive element; it also induces F-actin accumulation in NIH3T3 cell-line (Iguchi et al. 2008).

GPR56 has also been shown to interact with Gq/11 through specific tetraspanins, CD9 or CD81 which probably function as scaffolding proteins (Little, Helmer & Stipp, 2004). The study also showed that GPR65 can be internalised in response to phorbol ester (PMA).

GPR56 in Carcinogenesis and Metastasis
GPR56 have been found to over-express in various tumour tissues, involved in cell transformation through cell adhesion pathway (Ke et al. 2007).

However, in metastatic melanoma cells, GPR56 was found to express at low level; moreover, GPR56 exhibited an anti-metastatic effect by interacting with a cross-linking enzyme in the extracellular matrix, known as tissue transglutaminase (TG2), which binds to fibronectin and integrins to facilitate cell-adhesion (Xu et al. 2006; Xu & Hynes 2007).

Remarks:
For having a large impact on cell motility as well as with the demonstrated oncogenic potential, it makes sense for GPR56 to naturally possess an inbuilt antagonistic lid as a preventive measure for non-specific activity induced by any non-specific ligand. The ligand of GPR56 is yet unknown; suppose by an elimination process if it was possible to confirm that the receptor is indeed ligand-less, this mode of action via homodimerisation of the N-terminal regions would be a sole activation mechanism of this receptor. If so, the receptor function would be affected directly by the receptor density, and by the population of transmembrane or membrane-associated proteins present in the local environment. More detailed characterisations of GPR56 are awaited.

References
Chlang NY et al. J Biol Chem. 286(16):14215-25.
Iguchi T et al. 2008. J Biol Chem. 283(21): 14469–14478.
Jansen A & Andermann E. 2005. J Med Genet. 42:369–378.
Jin Z et al. 2007. Hum Mol Genet. 16(16):1972-85.
Ke N et al. 2007. Mol Cancer Ther. 6: 1840-1850.
Koirala S et al. 2009. J Neurosci. 29(23):7439-49.
Little KD, Helmer ME & Stipp CS. 2004. Mol Biol Cell. 15: 2375-2387.
Paavola KJ et al. 2011. J Biol Chem. June 27 in press.
Shima Y et al. 2007. Nat Neurosci 10(8): 963-969
Xu L et al. 2006. PNAS. 103(24): 9023–9028.
Xu L & Hynes RO 2007. Cell Cycle 6(2): 160‐165.