Allosteric modulation of adenosine A1 and cannabinoid 1 receptor signaling by G-peptides

Abstract

While allosteric modulation of GPCR signaling has gained prominence to address the need for receptor specificity, efforts have mainly focused on allosteric sites adjacent to the orthosteric ligand-binding pocket and lipophilic molecules that target transmembrane helices. In this study, we examined the allosteric influence of native peptides derived from the C-terminus of the Gα subunit (G-peptides) on signaling from two Gi-coupled receptors, adenosine A1 receptor (A₁ R) and cannabinoid receptor 1 (CB₁ ). We expressed A₁ R and CB₁ fusions with G-peptides derived from Gαs, Gαi, and Gαq in HEK 293 cells using systematic protein affinity strength modulation (SPASM) and monitored the impact on downstream signaling in the cell compared to a construct lacking G-peptides. We used agonists N⁶ -Cyclopentyladenosine (CPA) and 5'-N-Ethylcarboxamidoadenosine (NECA) for A₁ R and 2-Arachidonoylglycerol (2-AG) and WIN 55,212-2 mesylate (WN) for CB₁ . G-peptides derived from Gαi and Gαq enhance agonist-dependent cAMP inhibition, demonstrating their effect as positive allosteric modulators of Gi-coupled signaling. In contrast, both G-peptides suppress agonist-dependent IP₁ levels suggesting that they differentially function as negative allosteric modulators of Gq-coupled signaling. Taken together with our previous studies on Gs-coupled receptors, this study provides an extended model for the allosteric effects of G-peptides on GPCR signaling, and highlights their potential as probe molecules to enhance receptor specificity.

Keywords: CB1; GTP-binding proteins; N(6)-cyclopentyladenosine; SCH 442 416; adenosine A1; adenosine-5’-(N-ethylcarboxamide); cannabinoid; receptor; signal transduction.

FIGURE 1

Gα peptides differentially impact Gs and Gi signaling in Cannabinoid (CB₁) receptors. A, SPASM sensors for characterization of second messenger response. Schematics of the A₁R and CB₁ GPCR peptide sensors containing C‐terminal Gα peptides corresponding to s‐, i‐, or q‐ 5α helices separated with Gly‐Ser‐Gly (GSG)₄ linkers to ensure rotational freedom. The no‐pep (−) construct lacks the Gα C‐terminal peptide. Forskolin‐stimulated cAMP dose‐response curves of B, CB₁ agonist, 2‐Arachidonoylglycerol (2‐AG), and C, WIN 55,212‐2 mesylate (WN55212‐2) in a CB₁ no‐pep (−) sensor (representative curves from N = 2 independent biological replicates composed of ≥3 technical repeats each). cAMP levels shown in the absence (gray line) and presence (black line) of pertussis toxin (PTX) treatment. Ligands potentiate forskolin‐stimulated cAMP accumulation at 30 μmol/L, suggesting Gs bias (B and C, red dashed lines). 2‐AG and WIN 55,212‐2 mesylate (WN) inhibit forskolin‐stimulated cAMP at 50 nmol/L and 300 nmol/L, respectively, suggesting Gi bias (B and C, green dashed lines). cAMP levels of tethered CB₁ sensors after stimulation by forskolin and 30 μmol/L 2‐AG (D) or WN (E) (N = 5 independent biological replicates). F, summary of Gα peptide influence on Gs signaling and cAMP production in CB₁. Inhibition of forskolin‐stimulated cAMP by tethered CB₁ sensors after stimulation by 50 nmol/L 2‐AG (G) (N = 8 independent biological replicates) or 300 nmol/L WN (H) (N = 6 independent biological replicates). I, summary of Gα peptide influence on Gi signaling and cAMP inhibition in CB₁. GPCR‐Gα C‐terminal peptide sensors are compared with the no‐pep (−) control. Results are expressed as mean ± SE. ****P < .0001; *P < .05

FIGURE 2

Characterization of cAMP modulation in adenosine receptor (A₁R) by SPASM sensors. Forskolin‐stimulated cAMP dose‐response curves of (A), A₁R agonist, N⁶‐Cyclopentyladenosine (CPA). cAMP levels shown in the absence (gray line) and presence (black line) of pertussis toxin (PTX) treatment. 50 nmol/L CPA inhibits forskolin‐stimulated cAMP, suggesting Gi bias (A, green dashed line). B, Inhibition of forskolin‐stimulated cAMP by tethered A₁R peptide sensors after stimulation by 50 nmol/L CPA. C, Summary of Gα peptide influence on Gi signaling and cAMP inhibition. GPCR‐Gα C‐terminal peptide sensors are compared with the no‐pep (−) control. Results are expressed as mean ± SE. ***P < .001; **P < .01. N = 8 independent biological replicates

FIGURE 3

Gαq and Gαi peptides inhibit signaling through Gq. A, IP₁ dose‐response curve of A₁R agonists, CPA (gray lines) (representative curves from N = 2 independent biological replicates composed of ≥3 technical repeats each) and NECA (black line) (N = 3 technical repeats), with A₁R‐no‐pep (−) sensor. IP₁ levels shown in the absence (dark gray line) and presence (light gray line) of pertussis toxin (PTX) treatment. 100 μmol/L CPA or NECA stimulate IP₁ (A, blue dashed line). B, IP₁ signal from A₁R after stimulation by 100 μmol/L CPA (left) (N = 3 independent biological replicates) or NECA (right) (N = 4 independent biological replicates) in the presence of different Gα C‐terminal peptides compared to no‐pep (−) control. c, summary of Gα peptide influence on Gq signaling and IP₁ production in A₁R. D, IP₁ dose‐response curve of CB₁ agonists, 2‐AG (gray lines) (N = 5 independent biological replicates) and WN (black line) (N = 4 independent biological replicates), with CB₁‐no‐pep (−) sensor. IP₁ levels shown in the absence (dark gray line) and presence (light gray line) of pertussis toxin (PTX) treatment. 100 μmol/L 2‐AG or WN stimulate IP₁ (D, blue dashed line). E, IP₁ signal from CB₁ Gα C‐terminal peptide sensors after stimulation by 100 μmol/L 2‐AG (left) or WN (right) compared to no‐pep (−) control (N = 3 technical repeats). F, summary of Gα peptide influence on Gq signaling and IP₁ production in CB₁. Results are expressed as mean ± SE. ****P < .0001; ***P < .001; **P < .01