Development of novel protein scaffolds as alternatives to whole antibodies for imaging and therapy: status on discovery research and clinical validation.

Recent advances in combinatorial protein engineering have made it possible to develop antibody-based and non-Ig protein scaffolds that can potentially substitute for most whole antibody-associated properties. In theory, many different natural human protein backbones are suitable to be used as recombinant templates for engineering : antibody-derived scaffolds, carrier proteins that display a single binding interface, backbones that provide a rigid core structure suitable for grafting loops or protein scaffolds allowing the incorporation of variable loops in a favorable 3D configuration. In practice however, only a few have yielded the necessary properties to be translated into 'druggable Biologicals'. Amongst these properties, potential broad specificities towards any kind of target, ease of production, small size, good tolerability and low immunogenicity are essential and will be discussed in this review. Intellectual property is another key issue for the development of these protein scaffolds; although circumventing antibody-associated patents is often a major if not primary goal, clear advantages compared to whole antibodies must be presented to translate scaffold discovery into successful therapeutic drug candidates. In this review, a particular emphasis will be given to the most validated scaffolds that have reached the clinical development phase. Although the question of their immunogenicity is still open, preliminary clinical data do not point to any particular adverse immunogenic reactions although these are highly dependent on dosage, administration route and therapeutic indication. Finally, some of the emerging Biotechs developing protein scaffolds have been associated during the last two years with successful acquisitions by Big Pharmas and we will speak on the perspective positions of these proteins within the global Biologicals market.

Mutation in a protein kinase C phosphorylation site of the 5-HT1A receptor preferentially attenuates Ca2+ responses to partial as opposed to higher-efficacy 5-HT1A agonists.

The Thr(149)Ala mutation in a putative protein kinase C phosphorylation site of the 5-HT(1A) receptor's second intracellular loop has been shown to affect the closing of Ca(2+) channels and Ca(2+) mobilisation without interfering with the inhibitory cAMP pathway (Mol Pharmacol 52 (1997) 164). Here, the Ca(2+) responses for a series of 5-HT(1A) agonists were compared between the wild-type (wt) and mutant Thr(149)Ala 5-HT(1A) receptor as part of a fusion protein containing a G(alpha)(15) protein. Neither the mutation nor the fusion process modified the [(3)H]WAY 100635-based ligand binding profile of the fusion proteins as compared to the wt 5-HT(1A) receptor protein. Whereas at the wt 5-HT(1A) receptor, 5-HT induced a Ca(2+) response in CHO-K1 cells via endogenous G(i/o) proteins, the Ca(2+) response to 5-HT at the mutant Thr(149)Ala 5-HT(1A) receptor was fully dependent on either the co-expression or the fusion to a recombinant G(alpha)(15) protein. Buspirone, flesinoxan and 8-OH-DPAT produced a graded partial response (26 to 62%) at the wt 5-HT(1A):G(alpha)(15) fusion protein; F 13640, 5-CT and F 14679 behaved as higher-efficacy agonists with maximal Ca(2+) responses similar to 5-HT. The maximal Ca(2+) responses at the mutant Thr(149)Ala 5-HT(1A):G(alpha)(15) fusion protein were significantly attenuated for flesinoxan and 8-OH-DPAT (-45 and -36%, respectively); the response to the other 5-HT agonists was not significantly affected. A similar effect was observed upon treatment with phorbol 12-myristate 13-acetate at the Thr(149)Ala 5-HT(1A):G(alpha)(15) fusion protein. In conclusion, the amplitude of the Ca(2+) responses induced by partial, but not that to fuller 5-HT(1A) receptor agonists, is affected by the Thr(149)Ala mutation of the 5-HT(1A):G(alpha)(15) fusion protein.

Evidence for protean agonism of RX 831003 at alpha 2A-adrenoceptors by co-expression with different G alpha protein subunits.

Intrinsic properties of alpha(2) AR ligands were investigated by measuring two distinct signalling pathways via the alpha(2A) AR protein in CHO-K1 cells: (i) a Ca(2+) response mediated by a promiscuous G(alpha 15) protein; and (ii) a pertussis toxin-resistant [(35)S]GTP gamma S binding response mediated by a G(alpha o)Cys(351)Ile protein. The dexefaroxan analogue RX 831003 was virtually without intrinsic activity at the wt alpha(2A) AR via a G(alpha 15) protein, but induced a partial positive Ca(2+) response [pEC(50): 7.79 (0.17), E(max): 38+/-1% vs (-)-adrenaline] at the mutant Thr(373L)ys alpha(2A) AR. RX 831003 displayed a similar potency (pIC(50): 7.68 (0.21) for both the wt (E(max): -18+/-4%) and Thr(373)Lys alpha(2A) AR (E(max): -19+/-4%) inhibition of basal [(35)S]GTP gamma S binding via a G(alpha o)Cys(351)Ile protein. These data indicate that the alpha(2) AR ligand RX 831003 behaves as a protean agonist at the alpha(2A) AR and that its activity is highly dependent on the co-expressed G(alpha) protein subunit.

Pharmacological analysis of wild-type alpha, gamma and delta subtypes of the human peroxisome proliferator-activated receptor.

Three distinct peroxisome proliferator-activated receptor (PPAR) cDNAs were isolated from human brain RNA. Whereas the PPARdelta subtype perfectly matched the amino acid sequences reported in the Genbank database, several differences were found for the PPARalpha (Lys(123)Met, Ala(268)Val, Gly(296)Ala and Val(444)Ala) and PPARgamma2 (Met(8)Ile, Pro(9)Ala, Met(186)Ile, Pro(187)Ala and the deletion of a Gln(213) residue) subtypes. A pharmacological analysis was undertaken by co-expressing each PPAR subtype with a reporter plasmid containing a luciferase gene under the transcriptional control of a synthetic, triplicated PPAR response element in either HepG2 or Cos-7 cells. Whereas fenofibrate unselectively activated the PPARalpha and PPARdelta subtypes, the related BM-17.0744 compound was more potent and selective for PPARalpha. The thiazolidine dione derivatives rosiglitazone and pioglitazone were potent and selective PPARgamma2 agonists. L-165041, reported as a selective and potent PPARdelta ligand, displayed in this specified transactivation system, apart from its highly efficacious PPARdelta agonist activity, partial and full agonism at, respectively, PPARalpha and PPARgamma2 subtypes. In conclusion, transcriptional control of a luciferase gene by wild-type PPAR subtypes provides powerful recombinant assays to evaluate ligand's efficacy at these nuclear receptors.

Agonist-independent and -dependent oligomerization of dopamine D(2) receptors by fusion to fluorescent proteins.

Oligomerization of the short (D(2S)) and long (D(2L)) isoforms of the dopamine D(2) receptor was explored in transfected Cos-7 cells by their C-terminal fusion to either an enhanced cyan or enhanced yellow fluorescent protein (ECFP or EYFP) and the fluorescent fusion protein interaction was monitored by a fluorescence resonance energy transfer (FRET) assay. The pharmacological properties of the fluorescent fusion proteins, as measured by both displacement of [(3)H]nemonapride binding and agonist-mediated stimulation of [(35)S]GTPgammaS binding upon co-expression with a G(alphao)Cys(351)Ile protein, were not different from the respective wild-type D(2S) and D(2L) receptors. Co-expression of D2S:ECFP+D2S:EYFP in a 1:1 ratio and D2L:ECFP+D2L:EYFP in a 27:1 ratio resulted, respectively, in an increase of 26% and 16% in the EYFP-specific fluorescent signal. These data are consistent with a close proximity of both D(2S) and D(2L) receptor pairs of fluorescent fusion proteins in the absence of ligand. The agonist-independent D(2S) receptor oligomerization could be attenuated by co-expression with either a wild-type, non-fluorescent D(2S) or D(2L) receptor subtype, but not with a distinct beta(2)-adrenoceptor. Incubation with the agonist (-)-norpropylapomorphine dose-dependently (EC(50): 0.23+/-0.06 nM) increased the FRET signal for the co-expression of D2S:ECFP and D2S:EYFP, in support of agonist-dependent D(2S) receptor oligomerization. In conclusion, our data strongly suggest the occurrence of dopamine D(2) receptor oligomers in intact Cos-7 cells.

Reciprocal modulation of alpha(2A)-adrenoceptor and G(alpha o) protein states as determined by carboxy-terminal mutagenesis of a G(alpha o) protein.

The C-terminal portion of G(alpha) proteins plays a key role in their selective activation by cognate receptors. alpha(2A)-Adrenoceptors (alpha(2A)-ARs) can differentially inhibit or stimulate adenylyl cyclases by the activation of distinct G(i/o) and G(s) protein families. The implication of the C-terminal portion of G(alpha o) and G(alphas) proteins in their activation by alpha(2A)-ARs was analyzed by constructing mutant G(alpha o) proteins in which each of the last five amino acid positions were exchanged for those corresponding to a G(alphas) protein. Agonist-dependent, pertussis toxin-resistant binding of guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTP gamma S) revealed that the degree of positive efficacy of clonidine was highly dependent on the presence of a G(alpha o) protein-derived Gly amino acid as the -3 residue at the C-terminal portion of the protein. In contrast, antagonist properties for clonidine were observed for those mutants carrying a G(alphas) protein-derived Glu residue at this position. (-)-Epinephrine yielded almost similar maximal [(35)S]GTP gamma S binding responses, but its potency was decreased 22- to 150-fold at the -3 Glu containing mutant G(alpha o) proteins compared with those mutants containing a Gly. A 9- to 39-fold increase in the alpha(2A)-AR agonist equilibrium dissociation constants further reflected changes in the G(alpha) protein-induced alpha(2A)-AR state mediated by the specific Gly to Glu mutation in the C-terminal portion of the G(alpha o) protein. The present data emphasize the unique role of the -3 position at the G(alpha) protein C-terminal portion, independent of its surrounding peptidic environment, in constraining a structure favorable for activated receptor interaction and transmission of the mutation-induced conformational change from the G(alpha o) protein to the alpha(2A)-AR.

Analytical pharmacology of G protein-coupled receptors by stoichiometric expression of the receptor and G(alpha) protein subunits.

The description of a new family of recombinant proteins, which are constructed by the covalent fusion of the cDNA encoding a G protein-coupled receptor with that of a G(alpha) protein subunit, has recently been introduced as an original strategy to explore receptor pharmacology under defined experimental conditions. As such, a controlled 1:1 stoichiometry of receptor and G(alpha) protein expression can be achieved, as well as a forced spatial proximity to each other. Fusion proteins have been revealed as active at the receptor ligand binding level and functional at the G(alpha) protein and effector level. Insights on analytical pharmacological data are discussed for wild-type and mutant receptors interacting with a given G(alpha) protein subunit and different subtypes of either wild-type or mutant G(alpha) proteins activated by a single receptor subtype. A possible alteration of the receptor:G(alpha) protein selectivity may occur due either to the spatial proximity of both protein partners or to a constraint receptor state unable to accommodate to different G(alpha) protein states. Coactivation of endogenous G(alpha) proteins in host cells expressing a fusion protein has also been observed, but depends mainly on the coupling efficiency of the receptor and G(alpha) protein engaged in the fusion process. The ligand's apparent intrinsic activity has been shown to be either enhanced, attenuated, or unmodified when the functional responses of a fusion protein are compared to the coexpression of both fusion protein partners.

Real-time analysis of dopamine: antagonist interactions at recombinant human D2long receptor upon modulation of its activation state.

1. Antipsychotic drugs may mediate their therapeutic effects not only by preventing the binding of dopamine but also by decreasing the propensity of the dopamine receptor to assume an active R* state. Ligand-mediated activation and blockade of the recombinant human D(2long) receptor was investigated in CHO-K1 cells upon modulation of its R* state. 2. Both the Ala(371)Lys (A371K) and Thr(372)Arg (T372R) D2long receptor mutants could be activated in a ligand-dependent manner via a chimeric G(alphaq/o) protein, and more efficaciously so than with the promiscuous G(alpha15) protein. 3. Dopamine and partial agonists (E(max): lisuride >> (+)-UH 232 approximately bromerguride) displayed dissimilar Ca(2+) kinetic properties at wild-type and mutant receptors. A371K and T372R D2long receptor mutants demonstrated an attenuated and enhanced maximal response to these partial agonists, respectively. 4. Dopamine antagonists were unable to block the transient high-magnitude Ca(2+) phase at the wild-type D2long receptor upon simultaneous exposure to antagonist and dopamine, while full blockade of the low-magnitude Ca(2+) phase did occur at a later time (onset-time: haloperidol < bromerguride < (+)-butaclamol). A similar, though more efficacious, antagonist profile was also found at the A371K mutant receptor. Conversely, the blockade of the low-magnitude Ca(2+) phase was attenuated (haloperidol) or almost absent [(+)-butaclamol and bromerguride] at the T372R mutant receptor. 5. In conclusion, mutagenesis of the Ala(371) and Thr(372) positions affects in an opposite way the ligand-dependent activation and blockade of the D2long receptor. The observed attenuation of dopamine-mediated Ca(2+) signal generation with different decay-times may underlie distinct properties of the dopaminergic ligands.

Molecular cloning, pharmacological properties and tissue distribution of the porcine 5-HT(1B) receptor.

Using a combination of RT - PCR and inverse-PCR techniques, we amplified, cloned and sequenced a full-length porcine 5-HT(1B) receptor cDNA derived from porcine cerebral cortex. Sequence analysis revealed 1170 bp encoding an open reading frame of 390 amino acids showing a 95% similarity with the human 5-HT(1B) receptor. The recombinant porcine 5-HT(1B) cDNA was expressed in monkey Cos-7 cells and its pharmacological profile was determined by radioligand binding assay using [(3)H]-GR125743. The affinities of several agonists (L694247>ergotamine > or =5-carboxamidotryptamine=dihydroergotamine=5-HT>CP122638=zolmitriptan>sumatriptan) and putative antagonists (GR127935>methiothepin>SB224289>>ritanserin>ketanserin > or =BRL15572) correlated highly with those described for the recombinant human 5-HT(1B) receptor. In membranes obtained from cells co-expressing the porcine 5-HT(1B) receptor and a mutant G(alphao)Cys(351)Ile protein, 5-HT and zolmitriptan increased, while the 5-HT(1B) receptor antagonist SB224289 decreased basal [(35)S]-GTPgammaS binding, thus showing inverse agonism. The potency of zolmitriptan in the [(35)S]-GTPgammaS binding assay (pEC(50): 7.64+/-0.04) agreed with its affinity in displacing the antagonist [(3)H]-GR125743 (pK(i): 7.36+/-0.07). The 5-HT(1B) receptor mRNA was observed by RT-PCR in several blood vessels, cerebral cortex, cerebellum and trigeminal ganglion. In situ hybridization performed in frontal cerebral cortex sections revealed the expression of 5-HT(1B) receptor mRNA in pyramidal cells. In conclusion, we have cloned and established the amino acid sequence, ligand binding profile and location of the porcine 5-HT(1B) receptor. This information may be useful in exploring the role of 5-HT(1B) receptor in pathophysiological processes relevant for novel drug discovery in diseases such as migraine.

Analysis of ligand activation of alpha 2-adrenoceptor subtypes under conditions of equal G alpha protein stoichiometry.

Variations in the measurement of ligand's intrinsic activity between receptor subtypes is a common consequence of unequal receptor:G protein density ratios. We have investigated ligand activation at the alpha2-adrenoceptor (alpha2-AR) subtypes under defined expression conditions of one receptor molecule for one Galpha protein molecule using fusion proteins. Fusion between either a wt alpha2C AR or a mutant Thr382Lys alpha2C AR and a chimeric Galphaq/il protein displayed robust, transient (-)-adrenaline-mediated Ca2+ responses with similar potencies (pEC50: 7.78 and 7.66) and kinetic properties. A comparison of the intrinsic activities of alpha2 AR agonists found d-medetomidine to be the only compound with an efficacy similar to that of (-)-adrenaline. The Ca2+ responses as mediated by UK 14304, oxymetazoline and clonidine became more potent and efficacious at the Thr381Lys alpha2C AR, whereas the response as mediated by talipexole displayed a higher potency with an unaltered maximal response. Whereas only small differences in ligand's intrinsic activities between the wt alpha2A, alpha2B and alpha2C AR fusion proteins were observed with most ligands, oxymetazoline was virtually silent at the alpha2A AR while active as a partial and apparently full agonist at the alpha2C AR and alpha2B AR, respectively. The mutant alpha2 AR subtypes could be differentiated using the apparent positive efficacy of ligands that used to be defined as antagonists. The following rank order of maximal responses was observed for the Thr381Lys alpha2C AR: idazoxan approximately equals SKF 86466 > atipamezole >> dexefaroxan; Thr373Lys alpha2A AR: SKF 86466 > idazoxan = atipamezole > dexefaroxan; and Thr370Lys alpha2B AR: atipamezole > idazoxan dexefaroxan. RX 811059 (10 microM) was the only compound to be completely silent at both the wt and mutant alpha2 AR subtypes. In conclusion, silent alpha2 AR ligands are probably rare in these specified alpha2 AR systems. Most antagonists may actually possess partial agonist properties at the alpha2 AR subtypes, which are facilitated by the same mutation in the distal portion of their third intracellular loop.

Different regulation of RGS2 mRNA by haloperidol and clozapine.

Expression of RGS2 mRNA was transiently up-regulated in rat striatum (25% in the medial part and 50% in the lateral part), in contrast to cingulate cortex and lateral septum, 30 min after acute treatment with haloperidol (2 mg/kg, i.p.). This effect disappeared 24 hours post-drug treatment, similar to the acute and strong up-regulation (700% at 30 min) of c-fos mRNA. RGS3, 5, 6, 8 or 9 mRNAs were not affected. Clozapine (20 mg/kg, i.p.) at an approximately equivalent dose of D2 receptor occupancy in the striatum did not significantly affect RGS and c-fos mRNAs levels. We suggest that RGS2 mRNA expression may be differently up-regulated in a region-specific manner by antipsychotics.

Modulation of ligand responses by coupling of alpha(2A)-adrenoceptors to diverse G(alpha)-proteins.

The hypothesis that different signalling may be mediated via a single alpha(2A)-adrenoceptor (alpha(2A) AR) subtype was investigated by challenging alpha(2) AR ligands in combination with diverse recombinant wt, mutant, and chimeric G(alpha)-proteins. Possible coupling of alpha(2A) AR to endogenous G(alphai/o)-proteins in CHO-K1 cells was excluded by measuring pertussis toxin (PTX)-resistant [(35)S]GTPgammaS-binding responses as a common functional response to alpha(2A) AR activation. (-)-Adrenaline (10 microM) displayed the highest magnitude of [(35)S]GTPgammaS-binding response in the co-presence of a PTX-resistant G(alphao)Cys(351)Ile protein, whereas a decreased response was obtained with the mutant G(alphai1/2)-proteins. Replacement of the last six amino acids at the C-terminal portion of the G(alphao)-protein by the corresponding amino acid region of either the G(alphaz)-, G(alphas)-, G(alphaq)-, or G(alpha15)-protein and co-expression with the alpha(2A) AR resulted in similar maximal (-)-adrenaline-mediated [(35)S]GTPgammaS-binding responses with these chimeric G(alphao)-proteins. The ligands D-medetomidine, BHT 920 (6-allyl-5,6,7,8-tetrahydro-4H-thiazolo[4,5-d]azepin-2-ylamine) and (+)-RX 811059 (2-(2-ethoxy-2,3-dihydro-benzo[1,4]dioxin-2-yl)-4,5-dihydro-1H-imidazole) were weakly active or virtually inactive at the chimeric G(alphao/s)-, G(alphao/q)-, and G(alphao/15)-proteins in contrast to the G(alphao/z)-protein. Furthermore, combining the constitutively active mutant Thr(373)Lys alpha(2A) AR with these chimeric G(alphao)-proteins enhanced the apparent intrinsic activity of d-medetomidine and BHT 920. A similar observation was made using the corresponding fusion proteins, where the stoichiometry of the mutant alpha(2A) AR to the chimeric G(alphao)-protein was fixed at 1.0. These data indicate that a single ligand may display different magnitudes of activation at the alpha(2A) AR subtype coupled to chimeric G(alphao) proteins under controlled conditions of alpha(2A) AR: G(alphao)-protein expression.

Dynamic dopamine-antagonist interactions at recombinant human dopamine D(2short) receptor: dopamine-bound versus antagonist-bound receptor states.

Antipsychotic drugs comprise a wide range of structurally diverse compounds and are considered to be antagonists at dopamine D2 receptors. High-resolution kinetic analyses of their antagonist properties was performed by monitoring dynamic dopamine (DA)-antagonist interactions at the recombinant human dopamine D(2short) receptor. Time-dependent Ca2+ responses were measured following activation of a chimeric G(alphaq/o) protein in Chinese hamster ovary-K1 cells. DA (10 microM) induced a rapid, high-magnitude Ca2+ response (T(max) = 13.2 +/- 0.7 s) followed by a low-magnitude phase, which continued throughout the recorded time period (15 min). Of a large series of putative DA antagonists, (+)-UH 232 and bromerguride demonstrated positive, DA-like intrinsic activity at the presumably unoccupied, DA-free receptor; the other antagonists being silent. Antagonists differed in terms of their abilities to prevent the high-magnitude Ca2+ phase in the antagonist-bound receptor state, and to reverse the low-magnitude Ca2+ phase in the DA-bound state. The benzamide derivatives tropapride and nemonapride fully antagonized both the high- and low-magnitude Ca2+ response. Haloperidol, risperidone, and S 14066 also antagonized both responses but with a maximal effect of only 62 to 79%. Although preventing the high-magnitude response (85-95%), the further putative antagonists (+)-butaclamol (6%), bromerguride (27%), and domperidone (41%) reversed the low-magnitude response only weakly and partially. These Ca2+ data indicate that putative DA antagonists act differently, in particular, at the DA-bound D(2short) receptor.

Modulation of 5-HT(1A) receptor activation by its interaction with wild-type and mutant g(alphai3) proteins.

Constitutive and agonist-dependent activation of the recombinant human 5-HT(1A) receptor (RC: 2.1.5HT.01A) was investigated by co-expression with a rat G(alphai3) protein in Cos-7 cells. The interaction between the 5-HT(1A) receptor and rat G(alphai3) protein was modulated by substitution of the G(alphai3) protein site for pertussis toxin-catalysed ADP-ribosylation (cysteine(351)) by each of the natural amino acids. Enhanced basal [(35)S]GTPgammaS binding responses (+24 to +189%) were observed with the mutant G(alphai3) proteins containing at position 351 either a histidine, glutamine, serine, tyrosine or a nonpolar amino acid with the exception of a proline. With each of these mutant G(alphai3) proteins, spiperone (10 microM), but not WAY 100635 (10 microM), reduced (-22 to -60%, p<0.05) the enhanced basal [(35)S]GTPgammaS binding response. 5-HT (10 microM)-mediated [(35)S]GTPgammaS binding responses attained for some of the mutant G(alphai3)Cys(351) proteins (Phe, Met, Val and Ala) more than 300% of that obtained with the wt G(alphai3) protein. Similar results were also obtained with the prototypical 5-HT(1A) agonist 8-OH-DPAT and the partial agonist (-)-pindolol. Fusion proteins assembled from the 5-HT(1A) receptor and either the wt G(alphai3)Cys(351), mutant G(alphai3)Cys(351)Gly or G(alphai3)Cys(351)Ile protein displayed similar observations for these ligands as obtained by co-expression of the 5-HT(1A) receptor with each of these G(alphai3) proteins. Both the degree of 5-HT(1A) receptor activation by 8-OH-DPAT and (-)-pindolol, and its inhibition by spiperone, strongly correlate (r(2): 0.78-0.81) with the octanol/water partition coefficients of the mutated amino acid at position 351 of the G(alphai3) protein. The present data also suggest the wt G(alphai3) protein does not result in maximal activation of the 5-HT(1A) receptor by the agonists being investigated.

New substituted 1-(2,3-dihydrobenzo[1, 4]dioxin-2-ylmethyl)piperidin-4-yl derivatives with alpha(2)-adrenoceptor antagonist activity.

The emergence of a novel theory concerning the role of noradrenaline in the progression and the treatment of neurodegenerative diseases such as Parkinson's and Alzheimer's diseases has provided a new impetus toward the discovery of novel compounds acting at alpha(2)-adrenoceptors. A series of substituted 1-(2, 3-dihydrobenzo[1,4]dioxin-2-ylmethyl)piperidin-4-yl derivatives bearing an amide, urea, or imidazolidinone moiety was studied. Some members of this series of compounds proved to be potent alpha(2)-adrenoceptor antagonists with good selectivity versus alpha(1)-adrenergic and D(2)-dopamine receptors. Particular emphasis is given to compound 33g which displays potent alpha(2)-adrenoceptor binding affinity in vitro and central effects in vivo following oral administration.

Molecular cloning, sequence analysis and pharmacological properties of the porcine 5-HT(1D) receptor.

A cDNA encoding the full-length 5-HT(1D) receptor derived from porcine cerebral cortex was amplified, cloned and sequenced, using guinea-pig 5-HT(1D) receptor coding sequence oligonucleotide primers in reverse transcription-polymerase chain reaction (RT - PCR). The 5' and 3' ends of the porcine 5-HT(1D) receptor cDNA were verified by inverse PCR. Sequence analysis of porcine 5-HT(1D) receptor cDNA revealed an open reading frame of 1134 nucleotides encoding a polypeptide of 377 amino acids having 92% homology with the human 5-HT(1D) receptor and 88 - 90% homology with other species homologues. The porcine 5-HT(1D) receptor cDNA was further subcloned into a mammalian expression vector pcDNA3 and expressed in monkey Cos-7 cells. Radioligand binding assays using either [(3)H]-5-CT or [(3)H]-GR125743 on Cos-7 cell membranes showed that pK(i) values of 14 serotonin ligands were highly correlated with those obtained with the human 5-HT(1D) receptor. Nonetheless, a selective antagonist at the human 5-HT(1D) receptor, BRL15572, only poorly recognized the porcine homologue. Using membranes from cells co-expressing the porcine 5-HT(1D) receptor and rat G(alphail)Cys(351) Ile protein, it was shown that 5-HT and zolmitriptan increased, while ketanserin decreased basal [(35)S]-GTPgammaS binding. The potency of zolmitriptan in the [(35)S]-GTPgammaS binding assay (pEC(50): 8. 46+/-0.08) agreed with its affinity in displacing the radioligands [(3)H]-5-CT and [(3)H]-GR125743 (pK(i): 8.38+/-0.15 and 8.67+/-0.08, respectively). In conclusion, we have established the cDNA sequence and pharmacology of the cloned porcine 5-HT(1D) receptor. This information would be useful in exploring the role of divergent amino acid residues in the receptor-ligand interaction as well as the role of 5-HT(1D) receptor in pathophysiological processes relevant for novel drug discovery in diseases such as migraine.

Coupling of canine serotonin 5-HT(1B) and 5-HT(1D) receptor subtypes to the formation of inositol phosphates by dual interactions with endogenous G(i/o) and recombinant G(alpha15) proteins.

Molecular cloning and expression of canine (ca) serotonin 5-HT(1B) and ca 5-HT(1D) receptor subtypes showed that besides the lower binding affinity of ketanserin for the ca 5-HT(1D) receptor, the ligand binding profiles were similar to their human homologues. Site-directed mutagenesis studies suggest that a Gln(189) residue in the second extracellular loop of the ca 5-HT(1D) receptor may partially account for the lower binding affinity of ketanserin. The coupling of ca 5-HT(1B) and ca 5-HT(1D) receptor subtypes to the phospholipase C pathway was analyzed by measuring stimulation of inositol phosphate formation in COS-7 cells. Zolmitriptan potently stimulated (EC(50) = 4.9 nM) the inositol phosphate formation at ca 5-HT(1D) receptors in a fully pertussis toxin (PTX)-dependent manner, whereas only a weak PTX-resistant inositol phosphate response (26-29% at 10 microM zolmitriptan) could be detected for the ca 5-HT(1B) receptor at a similar expression level. In contrast, both ca 5-HT(1B) and ca 5-HT(1D) receptor subtypes yielded a similar maximal magnitude of inositol phosphate formation (300-340% at 10 microM zolmitriptan) upon co-expression with a mouse (m) G(alpha15) protein. PTX treatment and co-expression with a beta-adrenergic receptor kinase C-terminal polypeptide partially (20-46%) abolished the m G(alpha15) protein-dependent ca 5-HT(1B) and ca 5-HT(1D) receptor-mediated stimulation of inositol phosphate formation. This study suggests both 5-HT receptor subtypes can activate betagamma subunits of endogenous G(i/o) proteins besides their coupling to recombinant m G(alpha15) protein.

Agonist efficacy at the alpha2A-adrenoceptor:G alpha15 fusion protein: an analysis based on Ca2+ responses.

A fusion protein was constructed between the recombinant human alpha2A-adrenoceptor and a mouse G alpha15 protein to measure the efficacy of agonist-induced Ca2+ responses at a receptor:G alpha15 protein stoichiometry of 1. Activation of this fusion protein in CHO-K1 cells by (-)-adrenaline induced a time- and concentration-dependent (pEC50: 7.28+/-0.04) increase in the intracellular Ca2+ concentration. The magnitude of the Ca2+ response was related to the amount of the fusion protein and the number of surface alpha2A-adrenoceptor binding sites as estimated by [3H]RX 821002 binding. Whereas UK 14304 was as efficacious as (-)-adrenaline, the following ligands displayed partial agonist properties [Emax in percentage vs. (-)-adrenaline: d-medetomidine (76+/-3) > BHT 920 (53+/-3) > clonidine (39+/-4) >> oxymetazoline (10+/-1)]. This ligand activation profile was not affected over a 30-fold range of expression of the fusion protein in contrast to the observed enhancement of the partial agonists' maximal responses by co-expression of the alpha2A-adrenoceptor with increasing amounts of the G alpha15 protein. In conclusion, the fusion protein approach opens perspectives to quantify intrinsic activities of ligands under controlled experimental conditions of a fixed receptor:G alpha15 protein ratio of 1.

alpha(2A)-adrenoceptor: G(alphai1) protein-mediated pertussis toxin-resistant attenuation of G(s) coupling to the cyclic AMP pathway.

Fusion proteins were constructed between a recombinant human alpha(2A)-adrenoceptor and either a rat wild-type G(alphai1) or putative pertussis toxin-resistant form of the G(alphai1) protein (G(alphai1)Cys(351)Gly). [(3)H]2-[2-(2-Methoxy-1, 4-benzodioxanyl)]imidazoline hydrochloride (RX 821002) saturation binding experiments demonstrated that both fusion proteins were expressed at a similar level as the alpha(2A)-adrenoceptor co-expressed with either a wild-type G(alphai1) or mutant G(alphai1)Cys(351)Gly protein in COS-7 cells, and displayed a ligand binding profile similar to that for the alpha(2A)-adrenoceptor protein. In alpha(2A)-adrenoceptor-transfected COS-7 cells, 5-bromo-6-(2-imidazolin-2-yl-amino) quinoxaline tartrate (brimonidine, 10 microM) induced stimulation (151 +/- 28%) of adenosine 3',5'-cyclic monophosphate (cAMP) formation which was prevented by cholera toxin treatment, demonstrating a direct coupling of the alpha(2A)-adrenoceptor to an endogenous G(alphas) protein in COS-7 cells. Expression of either the wild-type G(alphai1) or mutant G(alphai1)Cys(351)Gly protein in co-expression or fusion with the alpha(2A)-adrenoceptor in COS-7 cells suppressed the brimonidine-induced stimulation of cAMP formation, both in the presence and absence of pertussis toxin pretreatment. Hence, the G(alphai1) protein apparently blocks the G(s)-coupled alpha(2A)-adrenoceptor-mediated pathway in a pertussis toxin-non-sensitive way.