title: Identification of distinct c-terminal domains of the Bombyx adipokinetic hormone receptor that are essential for receptor export, phosphorylation and internalization.
abstract: Neuropeptides of the adipokinetic hormone (AKH) family play important roles in insect hemolymph sugar homeostasis, larval lipolysis and storage-fat mobilization. Our previous studies have shown that the adipokinetic hormone receptor (AKHR), a Gs-coupled receptor, induces intracellular cAMP accumulation, calcium mobilization and ERK1/2 phosphorylation upon agonist stimulation. However, the underlying molecular mechanisms that regulate the internalization and desensitization of AKHR remain largely unknown. In the current study we made a construct to express AKHR fused with enhanced green fluorescent protein (EGFP) at its C-terminal end to further characterize AKHR internalization. In stable AKHR-EGFP-expressing HEK-293 cells, AKHR-EGFP was mainly localized at the plasma membrane and was rapidly internalized in a dose- and time-dependent manner via the clathrin-coated pit pathway upon agonist stimulation, and internalized receptors were slowly recovered to the cell surface after the removal of AKH peptides. The results derived from RNA interference and arrestin translocation demonstrated that G protein-coupled receptor kinase 2 and 5 (GRK2/5) and β-arrestin2 were involved in receptor phosphorylation and internalization. Furthermore, experiments using deletion and site-directed mutagenesis strategies identified the three residues (Thr356, Ser359 and Thr362) responsible for GRK-mediated phosphorylation and internalization and the C-terminal domain from residue-322 to residue-342 responsible for receptor export from ER. This is the first detailed investigation of the internalization and trafficking of insect G protein-coupled receptors.
authors.affiliation: Zhejiang Provincial Key Laboratory for Model Organisms, School of Life Sciences, Wenzhou Medical College, Wenzhou, Zhejiang, 325035, China.
authors.forename: Haishan
authors.initials: H
authors.lastname: Huang
authors.forename: Xiaoyan
authors.initials: X
authors.lastname: Deng
authors.forename: Xiaobai
authors.initials: X
authors.lastname: He
authors.forename: Wen
authors.initials: W
authors.lastname: Yang
authors.forename: Guo
authors.initials: G
authors.lastname: Li
authors.forename: Ying
authors.initials: Y
authors.lastname: Shi
authors.forename: Liangen
authors.initials: L
authors.lastname: Shi
authors.forename: Lijuan
authors.initials: L
authors.lastname: Mei
authors.forename: Jimin
authors.initials: J
authors.lastname: Gao
authors.forename: Naiming
authors.initials: N
authors.lastname: Zhou
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019390:Arrestins / Q000378:metabolism; D012831:Bombyx / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism*; D022163:Clathrin-Coated Vesicles / Q000378:metabolism; D003001:Cloning, Molecular; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005434:Flow Cytometry; D054768:G-Protein-Coupled Receptor Kinases / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D049452:Green Fluorescent Proteins / Q000378:metabolism; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D019476:Insect Proteins / Q000737:chemistry / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010766:Phosphorylation; D021381:Protein Transport; D034622:RNA Interference; D054417:Receptors, Adipokine / Q000737:chemistry / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017384:Sequence Deletion; D015398:Signal Transduction; D014162:Transfection; D000071557:beta-Arrestins
chemical_list: D019390:Arrestins; D019476:Insect Proteins; D054417:Receptors, Adipokine; D043562:Receptors, G-Protein-Coupled; D011993:Recombinant Fusion Proteins; D000071557:beta-Arrestins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D054768:G-Protein-Coupled Receptor Kinases; D002118:Calcium