Gavin J. Kilpatrick

7TM receptors: the splicing on the cake

Within a given family of seven transmembrane domain (7TM) receptors, functional diversity is most often afforded by the existence of multiple receptor subtypes, each encoded by a distinct gene. However, it is now clear that the existence of introns in genes encoding some members of a receptor family provides scope for additional diversity by virtue of splicing events that result in the formation of different receptor mRNAs and consequently distinct receptor isoforms. A large number of 7TM receptor splice variants have now been shown to exist. In this article, the current data on alternatively spliced variants for hormone and neurotransmitter 7TMs are reviewed, their potential physiological importance considered and some of the issues pertaining to the classification and nomenclature of receptor isoforms produced in this way are addressed.

A new functional isoform of the human CRF2 receptor for corticotropin-releasing factor

We have identified the human counterpart of the corticotropin-releasing factor receptor subtype 2beta. Its functional response to human urocortin was demonstrated after stable expression in HEK-293 cells. The receptor was also shown to bind sauvagine, corticotropin-releasing factor and urocortin. In contrast to rodents, the human CRF(2beta) receptor is only weakly expressed in heart and skeletal tissues, where the CRF(2alpha) isoform is predominant. Moreover, we have identified additional mRNAs of the CRF(2beta) type which are probably a consequence of aberrant splicing events.

Molecular biology of the CRH receptors— in the mood

Dysfunctioning of corticotropin-releasing hormone (CRH) and its receptors (CRH(1) and CRH(2)) has been linked to the development of stress-related disorders, such as mood and eating disorders. The molecular characterization of CRH(1) and CRH(2) receptors and their splice variants has generated detailed information on their pharmacology, tissue distribution and physiology. While mammalian CRH(1) receptors nonselectively bind CRH analogs, the ligand specificity of CRH(2) is narrower. CRH(1) receptors are predominantly expressed in the brain and pituitary, whereas CRH(2) receptor expression is limited to particular brain areas and to some peripheral organs. Molecular approaches to block CRH(1) receptor expression in the brain argue in favor of its involvement in the regulation of some aspects of the stress response. The CRH(2alpha) receptor may be more important for motivational types of behavior essential for survival, such as feeding and defense.(1)

Human CRF2 α and β splice variants: pharmacological characterization using radioligand binding and a luciferase gene expression assay

Abstract Corticotropin releasing factor (CRF) receptors belong to the super-family of G protein-coupled receptors. These receptors are classified into two subtypes (CRF1 and CRF2). Both receptors are positively coupled to adenylyl cyclase but they have a distinct pharmacology and distribution in brain. Two isoforms belonging to the CRF2 subtype receptors, CRF2α and CRF2β, have been identified in rat and man. The neuropeptides CRF and urocortin mediate their actions through this CRF G protein-coupled receptor family. In this report, we describe the pharmacological characterization of the recently identified hCRF2β receptor. We have used radioligand binding with [125I]-tyr0-sauvagine and a gene expression assay in which the firefly luciferase gene expression is under the control of cAMP responsive elements. Association kinetics of [125I]-tyr0-sauvagine binding to the hCRF2β receptor were monophasic while dissociation kinetics were biphasic, in agreement with the kinetics results obtained with the hCRF2α receptor. Saturation binding analysis revealed two affinity states in HEK 293 cells with binding parameters in accord with those determined kinetically and with parameters obtained with the hCRF2α receptor. A non-hydrolysable GTP analog, Gpp(NH)p, reduced the high affinity binding of [125I]-tyr0-sauvagine to both hCRF2 receptor isoforms in a similar manner. The rank order of potency of CRF agonist peptides in competition experiments was identical for both hCRF2 isoforms (urocortin>sauvagine>urotensin 1>r/hCRF>α-helical CRF(9–41)>oCRF). Similarly, agonist potency was similar for the two isoforms when studied using the luciferase gene reporter system. The peptide antagonist α-helical CRF(9–41) exhibited a non-competitive antagonism of urocortin-stimulated luciferase expression with both hCRF2 receptor isoforms. Taken together, these results indicate that the pharmacological profiles of the CRF2 splice variants are identical. This indicates that the region of the N-terminus that varies between the receptors is probably not important in the binding of peptide CRF receptor ligands or functional activation of the receptor.

Labelling of CRF1 and CRF2 receptors using the novel radioligand, [3H]-urocortin

The binding of the novel radioligand, [3H]-rat urocortin to homogenates of rat cerebellum and homogenates of cells stably transfected with the human CRF1, rat CRF2alpha and rat CRF2beta receptors was examined. In each case, specific reversible high affinity binding was observed (K[d]s between 0.18 and 0.31 nM). The density of sites was relatively low in the cerebellum (9 fmol/mg tissue) but high in the recombinant systems with expression levels of between 1.4 and 6.3 pmol/mg protein. Agents known to interact with CRF receptors potently competed for binding in each case. The pharmacological profile of binding to the recombinant receptors were consistent with data previously published using other radioligands. Thus, for the recombinant CRF1 receptor, binding was inhibited with similar affinity by Urocortin, sauvagine, Urotensin 1 and CRF. The non-peptidic CRF antagonists (e.g. CP 154,526 and SC 241) also potently inhibited binding. The CRF2alpha and CRF2beta receptor recombinant systems had a very similar pharmacological profile with a clear rank order of potency for the peptide ligands (Urocortin > Sauvagine > Urotensin 1 > CRF), whereas the non-peptide CRF receptor antagonists had no measurable affinity. The pharmacological profile of specific [3H]-urocortin binding to homogentates of rat cerebellum was consistent with specific labelling of a CRF1 receptor. We conclude that [3H]-urocortin is a useful tool for the study of CRF receptors with the advantages that a filtration assay can be used, all CRF receptors can be labelled with the same ligand and the benefits associated with the low energy emittor, 3H.

5-HT3 and 5-HT4 receptors in terminal regions of the mesolimbic system

In this brief review, we present the evidence for the regulation of the mesolimbic dopamine system by 5-HT3 and 5-HT4 receptors. A range of studies show good evidence that 5-HT3 receptor antagonists reduce raised mesolimbic dopamine activity by blocking 5-HT3 receptors in terminal parts of the mesolimbic dopamine system. Few studies have been conducted on the effects of 5-HT4 receptors on dopamine systems. However, it is clear that 5-HT4 receptors are present in relatively high density in areas of the brain that contain dopamine and preliminary studies show that 5-HT4 receptors may regulate the release of this transmitter.

Evidence for the abundant expression of arginine 185 containing human CRF2α receptors and the role of position 185 for receptor-ligand selectivity

The abundance of a histidine residue at position 185 (His(185)) of the human corticotropin-releasing factor (CRF) type 2 alpha receptor (hCRF(2alpha)) was investigated. His(185) has only been reported in hCRF(2); CRF(2) proteins from other species and all CRF(1) receptors encode an arginine (Arg(185)) at the corresponding position. Cloning of partial and full-length hCRF(2) cDNAs from a variety of neuronal and peripheral tissues revealed the existence of receptor molecules encoding Arg(185) only. Sequence analysis of the hCRF(2) gene verified the existence of Arg(185) also on genomic level. Full-length cDNAs encoding either the His(185) (R2H(185)) or the Arg(185) (R2R(185)) variants of hCRF(2alpha) were stably expressed in HEK293 cells and tested for ligand binding properties. In displacement studies R2H(185) and R2R(185) displayed a similar substrate specificity, human and rat urocortin, and the peptide antagonists astressin and alpha-helical CRF((9-41)) were bound with high affinity whereas human and ovine CRF were low-affinity ligands. Significant differences were observed for sauvagine and urotensin I, which bound with 3-fold (sauvagine) and 9-fold (urotensin I) higher affinity to R2R(185). These data indicate that hCRF(2), like all vertebrate CRF(1) and CRF(2) proteins encodes an arginine residue at the junction between extracellular domain 2 and transmembrane domain 3 and that this amino acid plays a role for the discrimination of some CRF peptide ligands.

Pharmacological characterisation of the recombinant human CRF binding protein using a simple assay

We present the pharmacological characterisation of the recombinant human corticotropin releasing factor binding protein (hCRF-BP) using a simple assay. In this assay we employed [3H]urocortin as the radioligand and, as a means to separate bound and free ligand, adsorption to activated charcoal. Using this method, approximately 60-70% of total binding was specific. Kinetic analysis revealed that association of specific [3H]urocortin binding was monophasic and slow and that the binding was irreversible. Saturation analysis showed a single saturable site of relatively high density (94 fmol per 10 microl of medium from cells transfected with the recombinant CRF binding protein). The apparent Kd for [3H]urocortin binding of 0.25 nM is similar to previously reported affinities of rat urocortin for hCRF-BP. A range of CRF-related peptides potently competed for specific [3H]urocortin binding. The rank order of potency of these agents was human/rat CRF = urotensin 1 > human urocortin > CRF6-33 > sauvagine > ovine CRF. The non-peptide CRF1 receptor antagonists CP 154,526 (N-butyl-N-[2,5-dimethyl-7-(2,4,6-trimethylphenyl)-7H-pyrrolo[2,3-d]p yri midin-4-yl]-N-ethylamine) and SC 241 ([3-(2-bromo-4-isopropyl-phenyl)-5-methyl-3H-[1,2,3]triazo lo[4,5-d]pyrimidin-7-yl]-bis-(2-methoxy-ethyl)-amine) were not active at the highest concentration tested (10(-6) M). We conclude that this is a simple and accurate assay for characterisation of the pharmacology of the recombinant CRF-BP. This assay should assist with further study of the pharmacology and function of the CRF-BP.

Urocortin: slower dissociation than corticotropin releasing factor from the CRF binding protein.

We report on a comparison of the kinetics of [125I][Tyr0]corticotropin releasing factor (CRF) and [125I][Tyr0]urocortin binding to the CRF binding protein (CRF-BP) at physiological temperature. The association rates of [125I][Tyr0]urocortin or [125I][Tyr0]CRF binding to the CRF-BP were similar. The half time of association for [125I][Tyr0]urocortin was 3.2 min and for [125I][Tyr0]CRF, 2.6 min. [125I][Tyr0]urocortin dissociated from the CRF-BP but the rate of dissociation was slower than for [125I][Tyr0]CRF. The half time for dissociation of [125I][Tyr0]urocortin was 131 min and for [125I][Tyr0]CRF, 64 min. This slower dissociation indicates that the CRF-BP may be more effective in clearing urocortin than CRF.