A key role for transmembrane prolines in calcitonin receptor-like receptor agonist binding and signalling:implications for family B G-protein-coupled receptors

Calcitonin receptor like-receptor is a family B G-protein coupled receptor (GPCR). It requires receptor activity modifying protein (RAMP) 1 to give a calcitonin gene-related peptide (CGRP) receptor. Little is known of how members of this receptor family function. Proline residues often form important kinks in alpha-helices. Therefore, all proline residues within the transmembrane helices of the receptor (Pro241, Pro244 in helix 4, Pro275 in helix 5, Pro321 and Pro331 in helix 6) were mutated to alanine. Pro241 Pro275, and Pro321 are highly conserved throughout all family B GPCRs. The binding of CGRP and its ability to stimulate cAMP production were investigated in mutant and wild-type receptors after transient transfection into COS-7 cells with RAMP1. The P321A mutation significantly decreased the pEC(50) for CGRP and reduced its affinity but did not change cell-surface expression. Antagonist binding [CGRP(8-37) and 1-piperidinecarboxamide N-[2-[[5amino-1-[[4-(4-pyridinyl)-1-piperazinyl]carbonyl]pentyl]amino]-1-[(3 5-dibromo-4-hydroxyphenyl)methyl]-2-oxoethyl]-4-(1,4-dihydro-2-oxo-3(2H)-quina zolinyl) (BIBN4096BS)] was little altered by the mutation. Adrenomedullin-mediated signaling was disrupted when P321A was coexpressed with RAMP1, RAMP2, or RAMP3. The P331A mutant produced a moderate reduction in CGRP binding and receptor activation. Mutation of the other residues had no effect on receptor function. Thus, Pro321 and Pro331 are required for agonist binding and receptor activation. Modeling suggested that Pro321 induces a bend in helix 6, bringing its C terminus near that of helix 3, as seen in many family A GPCRs. This is abolished in P321A. P321A-I325P predicted to restore this conformation, showed wild-type activation. Modeling can also rationalize the effects of transmembrane proline mutants previously reported for another family B GPCR, the VPAC(1) receptor.

Investigation into the biochemical changes in Tourette Syndrome with a potential for pharmacological manipulation

Kynurenine (KYN) is the first stable metabolite of the kynurenine pathway, which accounts for over 95% of tryptophan metabolism. Two previous studies by this research group reported elevated plasma KYN in Tourette syndrome (TS) patients when compared with age and sex matched controls and another study showed that KYN potentiated 5-HT2A-mediated head-shakes (HS) in rodents. These movements have been suggested to model tics in TS. This raised the questions how KYN acts in eliciting this response and whether it is an action of its own or of a further metabolite along the kynurenine pathway. In the liver, where most of the kynurenine pathway metabolism takes place under physiological conditions, the first and the rate limiting enzyme is tryptophan-dioxygenase (TDO) which can be induced by cortisol. In extrahepatic tissues the same step of the pathway is catalyzed by indoleamine-dioxygenase (IDO), which is induced by cytokines, predominantly interferon-y (INF-y). Plasma neopterin, which shows parallel increase with KYN following immune stimulation, was also found elevated in one of these studies positively correlating with KYN. In the present work animal studies suggested that KYN potentiates and quinolinic acid (QUINA) dose dependently inhibits the 5-HT2A-mediated HS response in mice. The potentiating effect seen with KYN was suggested to be an effect of KYN itself. Radioligand binding and phosphoinositide (PI) hydrolysis studies were done to explore the mechanisms by which kynurenine pathway metabolites could alter a 5-HT2A-receptor mediated response. None of the kynurenine pathway metabolites tested showed direct binding to 5-HT2A-receptors. PI hydrolysis studies with KYN and QUINA showed that KYN did not have any effect while QUINA inhibited 5-HT2A-mediated PI hydrolysis. Plasma cortisol determination in TS patients with elevated plasma KYN did not show elevated plasma cortisol levels, suggesting that the increase of plasma KYN in these TS patients is unlikely to be due to an increased TDO activity induced by increased cortisol. Attention deficit hyperactivity disorder (ADHD) is commonly associated with TS. Salivary cortisol detected in a group of children primarily affected with ADHD showed significantly lower salivary cortisol levels when compared with age and sex matched controls. Plasma tryptophan, KYN, neopterin, INF-y and KYN/tryptophan ratio and night-time urinary 6-sulphatoxymelatonin (aMT6s) excretion measured in a group of TS patients did not show any difference in their levels when compared with age and sex matched controls, but TS patients failed to show the expected positive correlation seen between plasma INF-y, neopterin and KYN and the negative correlation seen between plasma KYN and night-time urinary aMT6s excretion seen in healthy controls. The relevance of the kynurenine pathway, melatonin secretion and cortisol to Tourette Syndrome and associated conditions and the mechanism by which KYN and QUINA alter the 5-HT2A-receptor mediated HS response are discussed.