Creation of a Constitutively Activated State of the 5‐HT_2A Receptor by Site‐directed Mutagenesis: Revelation of Inverse Agonist Activity of Antagonists

Abstract

Constitutively active GPCR have revealed novel properties of drugs that exhibit classical competitive antagonism at the native forms of GPCR. These drugs reverse basal levels of constitutive activity, indicating that they have inverse agonist activity. We were interested in determining if competitive antagonists of the native 5-HT2A receptor, in particular, antipsychotic drugs, exhibit inverse agonist activity at the constitutively active 5-HT2A receptor. All of the drugs tested reduced basal IP production of constitutively active 5-HT2A receptors, indicating that they all exhibited inverse agonist activity. Risperidone and ketanserin produced the greatest inhibition of basal IP production resulting in a reduction of basal activity in the C322K mutant receptor of 82% and 80%, respectively. Antipsychotic drugs display inverse agonist activity, indicating that stabilization of the inactive conformation of the 5-HT2A receptor may be a key component of their mechanism of action.

Paradoxical trafficking and regulation of 5-HT2A receptors by agonists and antagonists

“…However, 5-HT 2A antagonists do not activate 5-HT 2A receptors as measured by any index of receptor activity (PI hydrolysis or arachidonic acid release) and do not induce cellular shape change [6]. In fact, clozapine has been demonstrated to be a potent antagonist with negative intrinsic activity at 5-HT 2A receptors [24]. Thus, based on the large number of studies reviewed here finding that 5-HT 2A antagonists can induce internalization and down-regulation, the current models of GPCR regulation must not be complete.…”

Endogenous Activation of Serotonin-2A Receptors Is Required for Respiratory Rhythm GenerationIn Vitro

“…This has been shown for a natural isoform of the 5-HT 2C receptor (Rauser et al, 2001). Indeed, ketanserine can reduce the activity of constitutively activated 5-HT 2A receptors, acting as an inverse agonist (Egan et al, 1998). The 5-HT 2 receptors are known to be linked to the G q family of G-proteins, and subsequent activation of phospholipase C causes an increase in intracellular calcium and the activation of PKC (Martin and Humphrey, 1994;Barnes and Sharp, 1999;Rauser et al, 2001).…”

Site‐Directed Mutations in the Third Intracellular Loop of the Serotonin 5‐HT1A Receptor Alter G Protein Coupling from Gi to Gs in a Ligand‐Dependent Manner

“…Restriction enzymes and other molecular biology reagents were purchased from Promega and Boehringer Mannheim Biochemicals (Samama et al, 1993); ␣ 2 -adrenoceptor (Ren et al, 1993); 5-HT 1B serotonin (Pauwels et al, 1999); 5-HT 2A serotonin (Egan et al, 1998); 5-HT 2C serotonin (Herrick-Davis et al, 1997); ␣ 1B -adrenoceptor (Kjelsberg et al, 1992). The arrow (m2 muscarinic) indicates the position of an insertion of one to four alanines also leading to constitutively active receptors (Liu et al, 1996).…”