The protease-activated thrombin receptor-1 (PAR-1) can be activated by both the tethered ligand exposed by thrombin cleavage and a synthetic peptide having the tethered ligand sequence (thrombin receptor agonist peptide or TRAP). We conducted a mutational analysis of extracellular residues of the receptor potentially involved in interaction with both the tethered ligand and the soluble peptide agonist. Agonist-stimulated calcium efflux in X. laevis oocytes or inositol phosphate accumulation in COS-7 cells was used to assess receptor activation. We have also examined the binding of a radiolabeled TRAP for the wild-type and mutant PAR-1 receptors. Our results indicated that most of the mutations strongly affected TRAP-induced responses without significantly altering thrombin-induced responses or TRAP binding. Several point mutations and deletion of extracellular domains (DeltaEC3, DeltaNH3) drastically altered the ability of mutant receptors to respond to TRAP, but not to thrombin, and did not affect the affinity for the radiolabeled TRAP by these mutant receptors. Only mutations that disrupted the putative disulfide bond or substitution of multiple acidic residues in the second extracellular loop by alanine had a significant effect on both ligand binding and thrombin activation. These results suggest that although both agonists can activate PAR-1, there are profound differences in the ability of thrombin and TRAP to activate PAR-1. In addition, we have found PAR-1 mutants with the ability to dissociate receptor-specific binding from functional activity.

The human NMDAR2D subunit was cloned, and the pharmacological properties of receptors resulting from injection of transcripts encoding human NMDAR1A and NMDAR2D subunits in Xenopus oocytes were characterized by profiling NMDA receptor agonists and antagonists. We found that glutamate, NMDA, glycine, and d‐serine were significantly more potent on hNMDAR1A/2D than on hNMDAR1A/2A or hNMDAR1A/2B. Also, the potencies of NMDA and glycine were higher for hNMDAR1A/2D than for hNMDAR1A/2C. Ifenprodil was more potent at hNMDAR1A/2B than at hNMDAR1A/2D, whereas 5,7‐dichlorokynurenate was more potent at hNMDAR1A/2A than at hNMDAR1A/2D. As measured in transiently transfected human embryonic kidney 293 cells, the maximal inward current in the presence of external Mg2+ occurred at −40 mV, and full block was not observed at negative potentials. Kinetic measurements revealed that the higher affinity of hNMDAR1A/2D for both glutamate and glycine relative to hNMDAR1A/2A and hNMDA1A/2B can be explained by slower dissociation of each agonist from hNMDAR1A/2D. The hNMDAR1A/2D combination represents a pharmacologically and functionally distinct receptor subtype and may constitute a potentially important target for therapeutic agents active in the human CNS.

The cDNA encoding hmGluR6, appended with a 15-amino acid antibody epitope (1D4), was transiently transfected in COS-7 cells. The receptor was purified from COS cell membranes using an antibody affinity column. The purified receptor was then reconstituted into lipid vesicles, and its ability to activate either transducin, the rod photoreceptor-specific GTP-binding protein, or the ␣ subunit of G o was assayed in vitro using a guanosine 5-3-O-(thio)triphosphate binding assay. Activation of both transducin and G o was observed. The rate of G o activation was 18-fold greater than the rate of transducin activation. This indicates that the coupling of mGluR6 to G o is more efficient and suggests that G o may be involved in coupling to mGluR6 in ON-bipolar cells.

cDNAs encoding four isoforms of the human NMDA receptor (NMDAR) NMDAR2C (hNR2C-1, -2, -3, and -4) have been isolated and characterized. The overall identity of the deduced amino acid sequences of human and rat NR2C-1 is 89.0%. The sequences of the rat and human carboxyl termini (G1y 925-Va11236) are encoded by different exons and are only 71.5% homologous. In situ hybridization in human brain revealed the expression of the NR2C mRNA in the pontine reticular formation and lack of expression in substantia nigra pars compacta in contrast to the distribution pattern observed previously in rodent brain. The pharmacological properties of hNR1A/2C were determined by measuring agonist-induced inward currents in Xenopus oocytes and compared with those of other human NMDAR subtypes. Glycine, glutamate, and NMDA each discriminated between hNR1A/2C-1 and at least one of hNR1A/2A, hNR1A/2B, or hNR1A/2D subtypes. Among the antagonists tested, CGS 19755 did not significantly discriminate between any of the four subtypes, whereas 5,7-dichlorokynurenic acid distinguished between hNR1A/2C and hNR1A/2D. Immunoblot analysis of membranes isolated from HEK293 cells transiently transfected with cDNAs encoding hNR1A and each of the four NR2C isoforms indicated the formation of heteromeric complexes between hNR1A and all four hNR2C isoforms. HEK293 cells expressing hNR1A/ 2C-3 or hNR1A/2C-4 did not display agonist responses. In contrast, we observed an agonist-induced elevation of intracellular free calcium and whole-cell currents in cells expressing hNR1A/2C-1 or hNR1A/2C-2. There were no detectable differences in the macroscopic biophysical properties of hNR1A/2C-1 or hNR1A/2C-2. Key Words: Cloning -Isoforms-N-Methyl-o-aspartate-Brain distribution-Biophysical properties-Pharmacology. J. Neurochem. 71, 1953Neurochem. 71, -1968Neurochem. 71, (1998.There is evidence that NMDA receptors (NMDARs) are involved in the induction and maintenance of longterm potentiation, synaptic integration, synaptogenesis, and developmental structuring of the mammalian CNS (Morris, 1989;Bliss and Collingridge, 1993) and in some forms of neuronal excitotoxicity (for review, see Rothman and Olney, 1995). Five NMDAR subunit cDNAs (NR1, NR2A, NR2B, NR2C, NR2D) have been cloned from rat and mouse (for review, see Mon and Mishina, 1995). in addition, several splice variants of the NRI gene have been cloned. Recombinant NMDARs have been functionally expressed in several host systems, and their biophysical and pharmacological properties have been characterized in detail (for review, see Hollmann and Heinemann, 1994;Mon and Mishina, 1995). The human (h) hNRIA, hNR2A, hNR2B, hNR2C, and hNR2D cDNAs have also been cloned (Karp et al., 1993;Le Bourdellès et al., 1994;Adams et al., 1995;Hess et al., 1996Hess et al., , 1998Lin et al., 1996).In this study, we describe the isolation and characterization of four isoforms of human NMDAR2C cDNAs (hNR2C-1, -2, -3, and -4). We identified a sequence divergence between human and rat cDNAs in the carboxyl-terminal regio...