Creation, expression, and characterization of a constitutively active mutant of the human serotonin 5‐HT₆ receptor

Abstract

The serotonin 5-HT(6) receptor, a G-protein-coupled receptor, displays high affinity for antipsychotic, antidepressant, and psychotropic drugs. We created a constitutively active form of the human 5-HT(6) receptor in order to probe the molecular domains of receptor activation and to determine if inverse agonist activities of antipsychotic drugs contribute to their clinical profile. Previous studies from our laboratory support a critical role for the c-terminal region of the third intracellular loop (il3) in the activation of G(q)-coupled serotonin receptors. In the present study, PCR-based mutagenesis was used to mutate serine 267 (S6.34) in the c-terminal region of il3 to lysine (S267K). The native and S267K 5-HT(6) receptors were expressed in COS-7 cells to study the functional effects of the mutation. The S267K receptor shows 10-fold higher affinity for serotonin than the native receptor and demonstrates agonist-independent activity. Clozapine decreased the basal activity of the S267K receptor to vector control levels. Therefore, we can conclude that the S267K mutation renders the 5-HT(6) receptor constitutively active and that clozapine is an inverse agonist at the mutant 5-HT(6) receptor. These results indicate that the c-terminal region of il3 of the G(s)-coupled 5-HT(6) receptor is a key domain for G-protein coupling, similar to the G(q)-coupled 5-HT receptors. The inverse agonist action of clozapine indicates that drugs displaying competitive antagonist activity at native 5-HT(6) receptors may display inverse agonist activity at the constitutively activated form of the receptor.

Physical interaction between neurofibromin and serotonin 5-HT 6 receptor promotes receptor constitutive activity

“…A mong 14 serotonin [5 hydroxytryptamine (5-HT)] receptor subtypes, the 5-HT 6 receptor has emerged as a promising target for the treatment of cognitive impairment associated with several neuropsychiatric disorders, including Alzheimer's disease and schizophrenia: 5-HT 6 receptor antagonists consistently enhance mnemonic performance in a broad range of procedures in rodents, and there is preliminary evidence for procognitive properties of 5-HT 6 receptor antagonists and/or inverse agonists in humans (1-3).…”

“…The 5-HT 6 receptor is a Gs-coupled receptor that activates cAMP formation on agonist stimulation in several recombinant systems (4)(5)(6) as well as in native systems, such as primary neurons (7) and pig caudate membranes (8). In addition to its coupling to G proteins, the 5-HT 6 receptor interacts with the Src family tyrosine kinase Fyn (9), the Jun activation domain-binding protein 1 (10), and the microtubule-associated protein Map1b (11).…”

“…In addition to its coupling to G proteins, the 5-HT 6 receptor interacts with the Src family tyrosine kinase Fyn (9), the Jun activation domain-binding protein 1 (10), and the microtubule-associated protein Map1b (11). The 5-HT 6 receptor also recruits the mammalian Target of Rapamycin (mTOR) Complex 1, and receptor-operated activation of mTOR signaling in prefrontal cortex (PFC) mediates its deleterious influence on cognition (12).…”

“…In addition to its coupling to G proteins, the 5-HT 6 receptor interacts with the Src family tyrosine kinase Fyn (9), the Jun activation domain-binding protein 1 (10), and the microtubule-associated protein Map1b (11). The 5-HT 6 receptor also recruits the mammalian Target of Rapamycin (mTOR) Complex 1, and receptor-operated activation of mTOR signaling in prefrontal cortex (PFC) mediates its deleterious influence on cognition (12). Furthermore, 5-HT 6 receptors associate with and activate Cyclin-dependent kinase 5 (Cdk5) in an agonist-independent manner through mechanisms involving receptor phosphorylation by associated Cdk5 to promote migration of neurons and neurite growth (13,14).…”

“…Constitutive activity of 5-HT 6 receptor was also established at Gs signaling in recombinant cells overexpressing WT or mutant receptors (5,6), but the underlying mechanism remains to be established. In light of recent evidence indicating that G protein-coupled receptor (GPCR) constitutive activity can be modulated by G protein-coupled receptor-interacting proteins (GIPs) (15), we focused on neurofibromin, another 5-HT 6 receptor partner known to be involved in adenylyl cyclase activation by various GPCRs (12,16).…”