Post-translational modifications of the apelin receptor regulate its functional expression

Toshihiko Kinjo; Shun Ebisawa; Tatsuya Nokubo; Mifu Hashimoto; Takonori Yamada; Michiko Oshio; Ruka Nakamura; Kyosuke Uno; Nobuyuki Kuramoto; Toshihiko Kinjo; Shun Ebisawa; Tatsuya Nokubo; Mifu Hashimoto; Takonori Yamada; Michiko Oshio; Ruka Nakamura; Kyosuke Uno; Nobuyuki Kuramoto

doi:10.3934/Neuroscience.2023022

AIMS Neuroscience

2023, Volume 10, Issue 4: 282-299. doi: 10.3934/Neuroscience.2023022

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Research article

Post-translational modifications of the apelin receptor regulate its functional expression

Laboratory of Molecular Pharmacology, Faculty of Pharmaceutical Sciences, Setsunan University, Hirakata, Osaka 573-0101, Japan

Received: 14 July 2023 Revised: 24 October 2023 Accepted: 26 October 2023 Published: 31 October 2023

Post-translational modifications (PTMs) are protein modifications that occur after protein biosynthesis, playing a crucial role in regulating protein function. They are involved in the functional expression of G-protein-coupled receptors (GPCRs), as well as intracellular and secretory protein signaling. Here, we aimed to investigate the PTMs of the apelin receptor (APLNR), a GPCR and their potential influence on the receptor's function. In an in vitro experiment using HEK cells, we only observed glycosylation as a PTM of the APLNR and ineffective receptor signaling by the agonist, (Pyr¹)-apelin-13. In contrast, when analyzing mouse spinal cord, we detected glycosylation and other PTMs, excluding isopeptidation. This suggests that additional PTMs are involved in the functional expression of the APLNR in vitro. In summary, these findings suggest that the APLNR in vivo requires multiple PTMs for functional expression. To comprehensively understand the pharmacological effects of the APLNR, it is essential to establish an in vitro system that adequately replicates the receptor's PTM profile. Nonetheless, it is crucial to overcome the challenge of heat-sensitive proteolysis in APLNR studies. By elucidating the regulation of PTMs, further research has the potential to advance the analysis and pharmacological studies of both the apelin/APLNR system and GPCR signal modulation.
- apelin/apelin receptor system,
- neurobiology,
- post-translational modifications,
- functional expression,
- glycosylation,
- ubiquitination
Citation: Toshihiko Kinjo, Shun Ebisawa, Tatsuya Nokubo, Mifu Hashimoto, Takonori Yamada, Michiko Oshio, Ruka Nakamura, Kyosuke Uno, Nobuyuki Kuramoto. Post-translational modifications of the apelin receptor regulate its functional expression[J]. AIMS Neuroscience, 2023, 10(4): 282-299. doi: 10.3934/Neuroscience.2023022

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Abstract

Post-translational modifications (PTMs) are protein modifications that occur after protein biosynthesis, playing a crucial role in regulating protein function. They are involved in the functional expression of G-protein-coupled receptors (GPCRs), as well as intracellular and secretory protein signaling. Here, we aimed to investigate the PTMs of the apelin receptor (APLNR), a GPCR and their potential influence on the receptor's function. In an in vitro experiment using HEK cells, we only observed glycosylation as a PTM of the APLNR and ineffective receptor signaling by the agonist, (Pyr¹)-apelin-13. In contrast, when analyzing mouse spinal cord, we detected glycosylation and other PTMs, excluding isopeptidation. This suggests that additional PTMs are involved in the functional expression of the APLNR in vitro. In summary, these findings suggest that the APLNR in vivo requires multiple PTMs for functional expression. To comprehensively understand the pharmacological effects of the APLNR, it is essential to establish an in vitro system that adequately replicates the receptor's PTM profile. Nonetheless, it is crucial to overcome the challenge of heat-sensitive proteolysis in APLNR studies. By elucidating the regulation of PTMs, further research has the potential to advance the analysis and pharmacological studies of both the apelin/APLNR system and GPCR signal modulation.

Abbreviations

AKT

protein kinase B

APLNR

apelin receptor

ATG

autophagy-related protein

cAMP

cyclic adenosine monophosphate

ERK

extracellular signal regulated kinase

FAT 10

F locus adjacent transcript 10

FSK

forskolin

GFP

green Fluorescent Protein

GPCR

G-protein coupled receptor

GPI

glycosylphosphatidylinositol

Gpr 44

G protein-coupled receptor 44

HEK

human embryonic kidney

ISG 15

interferon-stimulated gene 15

LPA 1

lysophosphatidic acid receptor 1

MAPK

mitogen-activated protein kinase

mTOR

mammalian target of rapamycin

NEDD

neural-precursor-cell-expressed developmentally down-regulated protein

NMDA

N-methyl-D-aspartate

PAR-1

protease-activated receptor 1

PDK1

phosphoinositide-dependent protein kinase 1

PI3K

phosphoinositide 3-kinase

PKA

protein kinase A

PTM

post-translational modification

SDS-PAGE

sodium dodecyl sulfate polyacrylamide gel electrophoresis

SUMO

small ubiquitin-like modifier

UFM 1

ubiquitin-fold modifier

URM 1

ubiquitin-related modifier-1

Acknowledgments

This work was supported by the Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (JSPS), Ministry of Education, Culture, Sports, Science and Technology of Japan (Grant Number JP21K06681).

Conflict of interest

The authors declare no conflict of interest.

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