Constitutive signal bias mediated by the human GHRHR splice variant 1

Abstract

Alternative splicing of G protein-coupled receptors has been observed, but their functions are largely unknown. Here, we report that a splice variant (SV1) of the human growth hormone-releasing hormone receptor (GHRHR) is capable of transducing biased signal. Differing only at the receptor N terminus, GHRHR predominantly activates G_s while SV1 selectively couples to β-arrestins. Based on the cryogenic electron microscopy structures of SV1 in the apo state or GHRH-bound state in complex with the G_s protein, molecular dynamics simulations reveal that the N termini of GHRHR and SV1 differentiate the downstream signaling pathways, G_s versus β-arrestins. As suggested by mutagenesis and functional studies, it appears that GHRH-elicited signal bias toward β-arrestin recruitment is constitutively mediated by SV1. The level of SV1 expression in prostate cancer cells is also positively correlated with ERK1/2 phosphorylation but negatively correlated with cAMP response. Our findings imply that constitutive signal bias may be a mechanism that ensures cancer cell proliferation.

Keywords: cancer; cell proliferation; class B1 GPCR; receptor bias.

Fig. 1.

GHRH-induced G_s protein coupling and cAMP signaling mediated by GHRHR and SV1. (A) GHRH-induced conformational changes in the trimeric G_s protein. Concentration-response curves are expressed as AUC across the time-course response curve (0 to 25 min) for each concentration and normalized to GHRHR. (B and C) Concentration-response curves of cAMP accumulation at GHRHR and SV1. Comparison of SV1 with full-length or truncated GHRHR that lacks ECD or the first 89 residues (B). Comparison of SV1 with various N terminus truncated GHRHRs (C). (D) Expression of GHRHR and SV1 in prostate cancer cell lines. Protein levels were estimated as relative intensity (RI) compared with β-tubulin (loading control). (E) Concentration-response curves of cAMP accumulation in prostate cancer cells. Data shown are means ± SEM of at least three independent experiments (n = 3 to 5) performed in quadruplicate. Δ, truncation; *P < 0.05; **P < 0.01.

Fig. 2.

GHRH-induced β-arrestin recruitment and pERK1/2 signaling mediated by GHRHR and SV1. (A and B) β-arrestin recruitment by GHRHR and SV1. Comparison of SV1 with full-length or truncated GHRHR that lacks the entire ECD or the first 89 residues (A). Comparison of SV1 and various N terminus truncated GHRHRs. The assay was initiated by 250 μM GHRH (B). The response was calculated as AUC across the full kinetic trace. *P < 0.01 and **P < 0.001 compared with GHRHR; Δ, truncation. (C and D) Time-course of ERK1/2 activation. The assay was initiated by 1 μM GHRH and inhibition was achieved by 4 μM MIA-602 in HEK293T cells expressing GHRHR or SV1 (C) and prostate cancer cell lines (D). Data shown are means ± SEM of at least four independent experiments (n = 4 to 6) performed in duplicate. *P < 0.05; **P < 0.01.

Fig. 3.

Overall structures of GHRH–SV1–Gα_s and apo SV1–Gα_s complexes. (A) Orthogonal views of the density map (Left) and the model (Right) for the GHRH–SV1–Gα_s–Nb35 complex. SV1, GHRH, Gα_s, Gβ, Gγ, and Nb35 are colored cornflower blue, gold, light green, salmon, cyan, and gray, respectively. (B) Orthogonal views of the density map (Left) and the model (Right) for the apo SV1–Gα_s–Nb35 complex. SV1, Gα_s, Gβ, Gγ, and Nb35 are colored slate blue, orange, salmon, cyan, and gray, respectively. The structures are shown in cartoon representation.

Fig. 4.

Structural comparison between SV1 and GHRHR. (A) Comparison between the cryo-EM structures of GHRH–SV1–G_s and GHRH–GHRHR–G_s complexes. Receptors and GHRH are shown in cartoon: GHRHR is colored in green, SV1 in blue, and GHRH in wheat and yellow. G_s is omitted for clarity. (B) Detailed interaction between GHRH (yellow) and the ECD of GHRHR (green). Key residues are shown as sticks. (C) Detailed interaction between GHRH (wheat) and the peptide-binding pocket of SV1 (blue). Salt bridges and hydrogen bonds are shown as dashed lines. (D) Effects of mutations in the peptide-binding pocket of SV1 on cAMP accumulation. Data shown are means ± SEM of four independent experiments (n = 4) conducted in quadruplicate.

Fig. 5.

β-arrestin 1 binding to GHRHR and SV1. (A) Distinct ECD conformations of GHRHR and SV1 during simulations. Receptors and GHRH are shown in cartoon: GHRHR is colored in green, SV1 in blue, and GHRH in wheat. β-arrestin 1 is omitted for clarity. (B) Representative simulation snapshots from GHRHR (Left) and SV1 (Right) systems. Gray dashed lines split the different regions of a receptor. (C) A representative simulation snapshot showing key interactions between GHRH (wheat) and the ECD of GHRHR (green). Key residues are shown as sticks. (D) Representative simulation snapshots showing the extracellular interfaces of GHRHR (Left) and SV1 (Right). A salt bridge of GHRHR (green) is shown as a black dashed line. (E) Representative simulation snapshots showing the receptor cores of GHRHR (Left) and SV1 (Right). A hydrogen bond of GHRHR (green) is shown as a black dashed line. (F) Binding of β-arrestin 1 to GHRHR (green) and SV1 (blue) at the intracellular side in simulations. (G) β-arrestin 1 recruitment by GHRHR and its mutants. The assay was stimulated by 250 μM GHRH. Data shown are means ± SEM of at least three independent experiments (n = 3 to 6) performed in duplicate; *P < 0.05, **P < 0.01.