GPR83 engages endogenous peptides from two distinct precursors to elicit differential signaling

Abstract

PEN is an abundant neuropeptide that activates GPR83, a G protein-coupled receptor that is considered a novel therapeutic target due to its roles in regulation of feeding, reward, and anxiety-related behaviors. The major form of PEN in the brain is 22 residues in length. Previous studies have identified shorter forms of PEN in mouse brain and neuroendocrine cells; these shorter forms were named PEN18, PEN19 and PEN20, with the number reflecting the length of the peptide. The C-terminal five residues of PEN20 are identical to the C-terminus of a procholecystokinin (proCCK)-derived peptide, named proCCK56-62, that is present in mouse brain. ProCCK56-62 is highly conserved across species although it has no homology to the bioactive cholecystokinin domain. ProCCK56-62 and a longer form, proCCK56-63 were tested for their ability to engage GPR83. Both peptides bind GPR83 with high affinity, activate second messenger pathways, and induce ligand-mediated receptor endocytosis. Interestingly, the shorter PEN peptides, ProCC56-62, and ProCCK56-63 differentially activate signal transduction pathways. Whereas PEN22 and PEN20 facilitate receptor coupling to Gai, PEN18, PEN19 and ProCCK peptides facilitate coupling to Gas. Furthermore, the ProCCK peptides exhibit dose dependent Ga subtype selectivity in that they faciliate coupling to Gas at low concentrations and Gai at high concentrations. These data demonstrate that peptides derived from two distinct peptide precursors can differentially activate GPR83, and that GPR83 exhibits Ga subtype preference depending on the nature and concentration of the peptide. These results are consistent with the emerging idea that endogenous neuropeptides function as biased ligands. Significance Statement We found that peptides derived from proCCK bind and activate GPR83, a G protein-coupled receptor that is known to bind peptides derived from proSAAS. Different forms of the proCCK- and proSAAS-derived peptides show biased agonism, activating G_as or G_ai depending on the length of the peptide and/or its concentration.

Keywords: Biased agonism; G protein coupled signaling; G proteins; g protein-coupled receptors (GPCRS).

Fig. 1.

Schematic representation of peptides generated from the processing of proSAAS and proCCK. Processing of the proSAAS precursor at monobasic and dibasic sites yields Big SAAS, Big GAV, KEP, Little SAAS, Little GAV, PEN, Big LEN, and Little LEN. Mass spectrometric analysis detects the presence of PEN (i.e., PEN22) as well as shorter PEN peptides (PEN18, PEN19, PEN20) (Mzhavia et al., 2002; Fricker, 2010). Processing of proCCK yields CCK8, CCK12, CCK33, CCK39, and CCK58. In addition, mass spectrometric analyses detect the presence of CCK peptides containing the highly conserved LGALL motif that is present in PEN (Mzhavia et al., 2002; Fricker, 2010).

Fig. 2.

Binding and signaling by C-terminally truncated PEN peptides at CHO-GPR83. (A) Sequence of full-length PEN (PEN22) and of the C-terminally truncated peptides, PEN18, PEN19, and PEN20. (B) C-terminally truncated PEN peptides displace [¹²⁵I]-Tyr-rPEN binding (3 nM) to membranes (20 μg) from CHO cells expressing GPR83 (CHO-GPR83). Data represent mean plus or minus S.D. (n = 3 for PEN18, PEN19, and PEN20 and n = 6 for PEN22). (C) C-terminally truncated PEN peptide-mediated increases in [³⁵S]GTPγS binding to CHO-GPR83 membranes (20 μg). Data represent mean plus or minus S.D. (n = 6). (D) Modulation of adenylyl cyclase activity by C-terminally truncated PEN peptides in CHO-GPR83 membranes (2 μg). Data represent mean plus or minus S.D. (n = 3). (E) Effect of pertussis toxin (PTX) and cholera toxin (CTX) treatment on PEN18-, PEN19-, PEN20-, and PEN22-mediated inhibition of adenylyl cyclase activity. Data represent mean plus or minus S.D. (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001 (one-way ANOVA with Tukey’s multiple comparison). (F) C-terminally truncated PEN peptide-mediated changes in phospholipase C activity in CHO-GPR83 cell membranes. Data represent mean plus or minus S.D. (n = 6). (G) C-terminally truncated PEN peptide-mediated increases in intracellular Ca⁺² levels in CHO-GPR83 cells. Data represent mean plus or minus S.D. (n = 3). (H) Dose-dependent modulation of GPR83 endocytosis by C-terminally truncated PEN peptides. (I) Time course of GPR83 internalization by C-terminally truncated PEN peptides. Data (H-I) represent mean plus or minus S.D. (n = 3). n.s., not significant.

Fig. 3.

Binding and signaling by C-terminally truncated PEN peptides at striatal membranes. (A) C-terminally truncated PEN peptides displace [¹²⁵I]-Tyr-PEN binding (3 nM) to striatal membranes (20 μg). Data represent mean plus or minus S.D. (n = 3). (B and C) C-terminally truncated PEN peptide-mediated increases in [³⁵S]GTPγS binding to striatal membranes (20 μg) from wild-type (B and C) and GPR83 knockout (C) mice. Data represent mean plus or minus S.D. (n = 3). In (C), *P < 0.05; **P < 0.01; WT versus GPR83 KO; unpaired t test with Welch’s correction. (D and E) Modulation of adenylyl cyclase activity by C-terminally truncated PEN peptides in striatal membranes (2 μg) from WT (D and E) and GPR83 KO (E) mice. Data represent mean plus or minus S.D. (n = 3). In (E), *P < 0.05; **P < 0.01; WT versus GPR83 KO; unpaired t test with Welch’s correction. (F) C-terminally truncated PEN peptide-mediated changes in phospholipase C activity in striatal membranes (10 μg). Data represent mean plus or minus S.D. (n = 3).

Fig. 4.

Binding and signaling by proCCK-derived peptides at CHO-GPR83. (A) Sequence of proCCK-derived peptides that exhibit similarity to peptides derived from PEN 22 (PEN11-20 and PEN11-22). (B and C) ProCCK-derived peptides and related PEN peptides displace [¹²⁵I]-Tyr-PEN binding (3 nM) to membranes (20 μg) from CHO-GPR83 (B) and WT striatum (C). Data represent mean plus or minus S.D. (n = 6). (D) ProCCK-derived peptide- and related PEN peptide–mediated increases in [³⁵S]GTPγS binding to striatal membranes (20 μg). Data represent mean plus or minus S.D. (n = 6). (E) PEN11-20 and PEN11-22–mediated increase in adenylyl cyclase activity in CHO-GPR83 cell membranes is blocked by CTX but not by PTX. Data represent mean plus or minus S.D. (n = 5 for PEN11-20 and PEN11-22 and n = 3 for PEN22). **P < 0.01; ***P < 0.001 (one-way ANOVA with Tukey’s multiple comparison test). (F) GPR83 endocytosis by proCCK-derived peptides and related PEN peptides in CHO-GPR83 cells. Data represent mean plus or minus S.D. (n = 6) (G) Modulation of adenylyl cyclase activity by proCCK-derived peptides in CHO and in CHO-GPR83 membranes (2 μg). Data represent mean plus or minus S.D. (n = 6). (H and I) A combination of CTX and PTX is needed to block CCK56-63–mediated changes in adenylyl cyclase activity in CHO-GPR83 (H) and striatal (I) membranes. Data represent mean plus or minus S.D. (n = 4).

Fig. 5.

Characterization of GPR83, proSAAS, and proCCK brain distribution in mice. The data for the mRNA expression was obtained from Allen Brain Mouse Atlas (https://mouse.brain-map.org/experiment), which shows in situ hybridization of sagittal mouse brain sections probed with GPR83 (A), proCCK (B), and proSAAS (gene name Pcsk1n) (C). Bar graphs represent the average raw expression value for different brain regions; each dot represents a biologic replicate. D. Diagram showing potential projections of proCCK- and proSAAS-derived neuropeptide release into brain regions with high levels of GPR83 expression. Projections of proCCK neurons and the expression of proSAAS-derived peptides were obtained from previous studies (Burgunder and Young, 1988; Schiffmann and Vanderhaeghen, 1991; Morino et al., 1994; You et al., 1994; Pesini et al., 1998; Brezillon et al., 2001; Wang et al., 2001; Adams et al., 2003; Whissell et al., 2015; Lueptow et al., 2018; Nguyen et al., 2020). Solid pink arrows represent the proCCK neuronal projections to striatum and olfactory tubercle (orange), both of which express GPR83 mRNA. Dashed green lines represent projections to the striatum and olfactory tubercle that express proSAAS mRNA, and these are potential sources of PEN in the GPR83-rich brain regions. CB, cerebellum; CTXsp, cortical subplate; HPF, hippocampal formation; HY, hypothalamus; MB, midbrain; MY, medulla; OLF, olfactory area; P, pons; PAL, pallidum; STR, striatum; TH, thalamus.