Identification and functional characterization of two orphan G-protein-coupled receptors for adipokinetic hormones from silkworm Bombyx mori.
Adipokinetic hormones (AKHs) are the best studied insect neuropeptides with the function of mobilizing lipids and carbohydrates during energy-expensive activities and modulating fundamental physiological processes, such as sugar homeostasis, lipid metabolism, and reproduction. Three distinct cDNAs encoding the prepro-Bombyx AKH1-3 have been cloned and confirmed by mass spectrometric methods. Our previous research suggested the Bombyx AKH receptor is activated by AKH1 and AKH2 with high affinity but by AKH3 with quite low affinity. In this study, using stable functional expression of the receptors in HEK293 cells, we have now identified AKH3 as a specific ligand for two orphan G-protein-coupled receptors, and we therefore named them AKHR2a and AKHR2b, respectively. We demonstrated that both AKHR2a and AKHR2b were activated by AKH3 at high affinity and by AKH1 and AKH2 at low affinity, leading to an increase of intracellular cAMP levels and activation of ERK1/2 and receptor internalization, but they were not activated by Bombyx corazonin. Conversely, the Bombyx corazonin receptor was activated by corazonin but not by AKH1-3. Quantitative RT-PCR revealed that AKHR2a and AKHR2b were both highly expressed in the testis but were also detected at low levels in other tissues. These results will lead to a better understanding of the AKH/AKHR system in the regulation of fundamental physiological processes.
Institute of Biochemistry, College of Life Sciences, Zijingang Campus, Zhejiang University, Hangzhou, Zhejiang, 310058.
Ying
Y
Shi
Zhejiang Provincial Key Laboratory for Model Organisms, School of Life Sciences, Wenzhou Medical College, Wenzhou, Zhejiang, 325035.
Haishan
H
Huang
Institute of Biochemistry, College of Life Sciences, Zijingang Campus, Zhejiang University, Hangzhou, Zhejiang, 310058.
Xiaoyan
X
Deng
Institute of Biochemistry, College of Life Sciences, Zijingang Campus, Zhejiang University, Hangzhou, Zhejiang, 310058.
Xiaobai
X
He
College of Animal Sciences, Zijingang Campus, Zhejiang University, Hangzhou, Zhejiang, 310058, China.
Jingwen
J
Yang
Institute of Biochemistry, College of Life Sciences, Zijingang Campus, Zhejiang University, Hangzhou, Zhejiang, 310058.
Huipeng
H
Yang
College of Animal Sciences, Zijingang Campus, Zhejiang University, Hangzhou, Zhejiang, 310058, China.
Liangen
L
Shi
Institute of Biochemistry, College of Life Sciences, Zijingang Campus, Zhejiang University, Hangzhou, Zhejiang, 310058; Zhejiang Provincial Key Laboratory for Model Organisms, School of Life Sciences, Wenzhou Medical College, Wenzhou, Zhejiang, 325035.
Lijuan
L
Mei
Zhejiang Provincial Key Laboratory for Model Organisms, School of Life Sciences, Wenzhou Medical College, Wenzhou, Zhejiang, 325035. Electronic address: jimingao@yahoo.com.
Jimin
J
Gao
Institute of Biochemistry, College of Life Sciences, Zijingang Campus, Zhejiang University, Hangzhou, Zhejiang, 310058. Electronic address: znm2000@yahoo.com.
Naiming
N
Zhou
D000595:Amino Acid Sequence; D000818:Animals; D012831:Bombyx; D002460:Cell Line; D005434:Flow Cytometry / Q000379:methods; D057809:HEK293 Cells; D006706:Homeostasis; D006801:Humans; D007301:Insect Hormones / Q000737:chemistry* / Q000378:metabolism; D019476:Insect Proteins / Q000378:metabolism; D008024:Ligands; D008969:Molecular Sequence Data; D009479:Neuropeptides / Q000378:metabolism; D009842:Oligopeptides / Q000737:chemistry* / Q000378:metabolism; D010802:Phylogeny; D011761:Pyrrolidonecarboxylic Acid / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D014162:Transfection
D007301:Insect Hormones; D019476:Insect Proteins; D008024:Ligands; D009479:Neuropeptides; D009842:Oligopeptides; D043562:Receptors, G-Protein-Coupled; C044833:adipokinetic hormone; C060602:corazonin protein, insect; D011761:Pyrrolidonecarboxylic Acid