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 Gs 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 Gs protein, molecular dynamics simulations reveal that the N termini of GHRHR and SV1 differentiate the downstream signaling pathways, Gs 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.

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“…For example a N-terminal splice variant of GHRHR , which has been shown to alter the signalling properties (i.e. G s vs β-arrestins), is also predicted to mildly alter corresponding couplings ( Supplementary Table S9 ) ( 43 ).…”

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“…Strikingly, we detected little to no density for the ECD ( Figure 1a ), indicating that it is very mobile, and this was also reflected in the 3DVA where density for the ECD was absent even at low contour ( Video S1 ). This observation supports the hypothesis that class B1 GPCR ECDs are highly dynamic when they are not anchored by interaction of ligands, or other proteins such as receptor activity-modifying proteins (Josephs et al ., 2021), that bridge the TMD and ECD (Yang et al ., 2015; Cong et al ., 2021).…”

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“…MD simulations have been extensively used to study GPCR interactions with signaling molecules − as well as transducers in the cytosolic side. − For example, Park et al recently studied the neuropeptide Y and Y1 receptor, which is enriched in the brain and responsible for neurological processes such as food intake, anxiety, obesity, and cancer. , The cryo-EM structure shows the binding of neuropeptide Y to Y1 receptor, and MD simulations further reveal the stability and dynamics of the neuropeptide Y binding, providing a conformational ensemble of binding interactions (Figure A). Other studies have examined the GPCR-G protein binding and tried to derive a rationale for signaling through different G proteins or β-arrestin. − Zhao et al studied β2 adrenaline receptors and their differential binding interfaces with Gs, Gi, and β-arrestin 1 . Since the binding interface is different for each cellular transducer, the conformational rearrangement in the TM domain is also different, allowing signal transduction to happen.…”

“…GPCRs play a vital role in relaying information from neurotransmitters and hormone by transmitting the binding of these molecules from the extracellular side to signals on the intracellular side via binding with cellular transducers such as G protein or β-arrestin. MD simulations have been extensively used to study GPCR interactions with signaling molecules − as well as transducers in the cytosolic side. − For example, Park et al recently studied the neuropeptide Y and Y1 receptor, which is enriched in the brain and responsible for neurological processes such as food intake, anxiety, obesity, and cancer. , The cryo-EM structure shows the binding of neuropeptide Y to Y1 receptor, and MD simulations further reveal the stability and dynamics of the neuropeptide Y binding, providing a conformational ensemble of binding interactions (Figure A). Other studies have examined the GPCR-G protein binding and tried to derive a rationale for signaling through different G proteins or β-arrestin. − Zhao et al studied β2 adrenaline receptors and their differential binding interfaces with Gs, Gi, and β-arrestin 1 .…”