Targeting orphan G protein-coupled receptors for the treatment of diabetes and its complications: C-peptide and GPR146

Abstract

G protein-coupled receptors (GPCRs) are the most abundant receptor family encoded by the human genome and are the targets of a high percentage of drugs currently in use or in clinical trials for the treatment of diseases such as diabetes and its associated complications. Thus, orphan GPCRs, for which the ligand is unknown, represent an important untapped source of therapeutic potential for the treatment of many diseases. We have identified the previously orphan GPCR, GPR146, as the putative receptor of proinsulin C-peptide, which may prove to be an effective treatment for diabetes-associated complications. For example, we have found a potential role of C-peptide and GPR146 in regulating the function of the retinal pigment epithelium, a monolayer of cells in the retina that serves as part of the blood-retinal barrier and is disrupted in diabetic macular oedema. However, C-peptide signalling in this cell type appears to depend at least in part on extracellular glucose concentration and its interaction with insulin. In this review, we discuss the therapeutic potential of orphan GPCRs with a special focus on C-peptide and GPR146, including past and current strategies used to 'deorphanize' this diverse family of receptors, past successes and the inherent difficulties of this process.

Keywords: C-peptide; G protein-coupled receptor; GPR146; diabetes; diabetes-associated complications; orphan GPCR.

Fig. 1

The complexity of G protein-coupled receptor (GPCR) signaling. (A) Canonical GPCR signaling via a stimulatory G alpha protein. (B) Association with receptor activity-modifying proteins (RAMPs) (blue, green, and purple bars) changes the ligand specificity of some GPCRs. Downstream signaling cascades may be altered as well (represented by colored boxes). Some GPCRs form homodimers (C) or heterodimers with other GPCRs (D), which can change ligand and/or downstream signaling molecule specificity.

Fig. 2

Drug target discovery and receptor deorphanization strategies.

Fig. 3

Utilization of the deductive ligand–receptor matching strategy for the identification of C-peptide receptor candidates. C-peptide-responsive cell lines were screened for the expression of orphan G protein-coupled receptors (GPCRs). Orphan GPCRs expressed by all responsive cell lines were then knocked down individually using siRNA in KATOIII cells. Cells were treated with C-peptide, and C-peptide signaling was assessed. Knockdown of GPR146 resulted in loss of C-peptide-induced signaling, and thus was considered the best candidate for the C-peptide receptor.

Fig. 4

Co-localization of C-peptide and GPR146 on KATOIII cell membranes. KATOIIl cells were co-incubated with 1 nM C-peptide and 1 nM insulin and stained using antibodies against C-peptide (red) and GPR146 (green). Co-localized particles appear yellow. Representative images from three visual fields.

Fig. 5

C-peptide-induced cFos mRNA expression in the human retinal pigment epithelial cell line ARPE-19. ARPE-19 cells were incubated in either low (5.5 mM) or high (25 mM) glucose for 24 h prior to exposure to vehicle, 1 nM C-peptide, or 1 nM C-peptide with 1 nM insulin for 1 h. Cells were lysed, RNA isolated, and changes in cFos mRNA expression evaluated using quantitative PCR. **P < 0.01, ***P < 0.001 vs. low glucose, vehicle-treated cells; ^#P < 0.01 vs. low glucose, C-peptide-treated cells.

Fig. 6

C-peptide and retinal pigment epithelium (RPE) cell gene expression changes in a transdifferentiation assay. ARPE-19 cells were incubated in either low (5.5 mM) or high (25 mM) glucose for 30 days with or without 1 nM C-peptide. Medium +/− C-peptide was replenished weekly. To evaluate C-peptide-induced changes in gene expression, cells were lysed, RNA isolated, and changes in gene expression evaluated by quantitative PCR using primers specific against genes produced by RPE cells, including the tight junction protein zona occludens 1 (ZO-1), the VEGF receptor 2 (VEGFR2), the transcription factor orthodenticle homeobox 2 (OTX2), the transcription factor microphthalmia-associated transcription factor (MITF), which is involved in regulating RPE cell identity, retinaldehyde-binding protein 1 (RLBP-1), and pigment epithelium-derived factor (PEDF). Under low glucose conditions, C-peptide significantly altered the mRNA expression of several genes, including ZO-1, MITF, and RLBP-1. However, this effect was lost in cells cultured in high glucose, suggesting that C-peptide-induced changes in gene expression are dependent upon glucose concentration, perhaps reflecting an interaction with AMPK-mediated signaling cascades. *P ≤ 0.05, **P ≤ 0.01 versus vehicle-treated control.