title: RhoBTB3 interacts with the 5-HT7a receptor and inhibits its proteasomal degradation.
abstract: The 5-hydroxytryptamine (5-HT)7 receptor is the most recently identified serotonin receptor and is involved in a wide variety of central nervous system (CNS) functions, namely circadian rhythm, REM sleep, depression, thermoregulation, obsessive-compulsive disorder (OCD), anxiety, schizophrenia, epilepsy, nociception, migraine, sensation-seeking behavior, impulsivity, learning and memory. These numerous (patho)physiological processes of the CNS, in which the 5-HT7 receptor is involved, most likely reflect a diverse set of signaling pathways arising from this receptor. In order to reveal new interaction partners and possibly new signaling and/or trafficking pathways, we performed a yeast two-hybrid screening, using the C-terminal tail of the 5-HT7a receptor as bait and an adult-human brain cDNA library as prey. In this way we identified RhoBTB3 as a new interaction partner of the 5-HT7a receptor. By means of co-immunoprecipitation we were able to confirm the interaction between full length 5-HT7a receptor and RhoBTB3 in HEK293T cells. Subsequent domain mapping of this interaction revealed that not only the C-terminal tail, but also the third intracellular loop of the 5-HT7a receptor is involved. In addition, immunofluorescence microscopy showed clear co-localization between the 5-HT7a receptor and RhoBTB3 at the plasma membrane and in the endoplasmic reticulum. Despite the fact that RhoBTB3 has been shown to interact with Cul3, which in turn interacts with the E3 ubiquitin ligase, Roc1, we show here that RhoBTB3 neither recruits Cul3/Roc1 to the 5-HT7a receptor nor does it mediate ubiquitination of this receptor. Instead, we demonstrate that RhoBTB3 strongly inhibits proteasomal degradation of the 5-HT7a receptor.
authors.affiliation: Laboratory of Eukaryotic Gene Expression and Signal Transduction (LEGEST), Department of Physiology, Ghent University (UGent), K.L. Ledeganckstraat 35, Gent 9000, Belgium.
authors.forename: Anne
authors.initials: A
authors.lastname: Matthys
authors.forename: Kathleen
authors.initials: K
authors.lastname: Van Craenenbroeck
authors.forename: Béatrice
authors.initials: B
authors.lastname: Lintermans
authors.forename: Guy
authors.initials: G
authors.lastname: Haegeman
authors.forename: Peter
authors.initials: P
authors.lastname: Vanhoenacker
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000378:metabolism; D044842:Cullin Proteins / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D007976:Leupeptins / Q000494:pharmacology; D008969:Molecular Sequence Data; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D061988:Proteasome Inhibitors; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism; D059748:Proteolysis; D011985:Receptors, Serotonin / Q000737:chemistry / Q000378:metabolism*; D020798:Two-Hybrid System Techniques; D020741:rho GTP-Binding Proteins / Q000378:metabolism*
chemical_list: C503645:CUL3 protein, human; D044842:Cullin Proteins; D007976:Leupeptins; D061988:Proteasome Inhibitors; D020033:Protein Isoforms; D011985:Receptors, Serotonin; C104425:serotonin 7 receptor; D046988:Proteasome Endopeptidase Complex; C541346:RhoBTB3 protein, human; D020741:rho GTP-Binding Proteins; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde