Differential coupling of dopaminergic D2 receptors expressed in different cell types. Stimulation of phosphatidylinositol 4,5-bisphosphate hydrolysis in LtK- fibroblasts, hyperpolarization, and cytosolic-free Ca2+ concentration decrease in GH4C1 cells

Dopaminergic D2 receptors are widely regarded as typical inhibitory receptors, as they both inhibit adenylyl cyclase and decrease the cytosolic free Ca2+ concentration ([Ca2+]i) by activating K+ channels. A D2 receptor has recently been cloned (Bunzow, J. R., Van Tol, H. H. M., Grandy, D. K., Albert, P., Salon, J., Christie, M. D., Machida, C. A., Neve, K. A., and Civelli, O. (1988) Nature 336, 783-787) and expressed in two different cell lines, pituitary GH4C1 cells and Ltk- fibroblasts, where it has been shown to induce inhibition of adenylyl cyclase. We have investigated the additional effector systems coupled to this receptor. The responses observed in the two cells lines, which express similar levels of receptors (0.5-1 x 10(5)/cell), were surprisingly different. In GH4C1 cells D2 receptors failed to affect phosphoinositide hydrolysis and induced a decrease of [Ca2+]i. This latter effect appears to be mediated by hyperpolarization, most likely due to the activation of K+ channels. In striking contrast, in Ltk- fibroblasts the D2 receptor induced a rapid stimulation of inositol(1,4,5)-trisphosphate (+73% at 15 s) followed by the other inositol phosphates, and an immediate increase of [Ca2+]i due to both Ca2+ mobilization from internal stores and influx from the extracellular medium. In both GH4C1 and Ltk- cells, the D2 receptor response was mediated by G protein(s) sensitive to pertussis toxin. The increases of inositol trisphosphate and [Ca2+]i observed in Ltk- cells required dopamine concentrations only slightly higher than those inhibiting adenylyl cyclase (EG50 = 25, 29, and 11 nM, respectively) and were comparable in magnitude to the responses induced by the endogenous stimulatory receptor agonists, thrombin and ATP. The results demonstrate that in certain cells D2 receptors are efficiently coupled to the stimulation of phosphoinositide hydrolysis. The nature of receptor responses appears therefore to depend on the specific properties not only of the receptor molecule but also of the cell type in which it is expressed.     

The E295K DNA polymerase beta gastric cancer-associated variant interferes with base excision repair and induces cellular transformation

Approximately 30% of human tumors examined for mutations in polymerase beta (pol beta) appear to express pol beta variant proteins (D. Starcevic, S. Dalal, and J. B. Sweasy, Cell Cycle 3:998-1001, 2004). Many of these variants result from a single amino acid substitution. We have previously shown that the K289M and I260M colon and prostate cancer variants, respectively, induce cellular transformation most likely due to sequence-specific mutator activity (S. Dalal et al., Biochemistry 44:15664-15673, 2005; T. Lang et al., Proc. Natl. Acad. Sci. USA 101:6074-6079, 2004; J. B. Sweasy et al., Proc. Natl. Acad. Sci. USA 102:14350-14355, 2005). In the work described here, we show that the E295K gastric carcinoma pol beta variant acts in a dominant-negative manner by interfering with base excision repair. This leads to an increase in sister chromatid exchanges. Expression of the E295K variant also induces cellular transformation. Our data suggest that unfilled gaps are channeled into a homology-directed repair pathway that could lead to genomic instability. The results indicate that base excision repair is critical for maintaining genome stability and could therefore be a tumor suppressor mechanism.     

Evidence for hybrid rodent and human insulin receptors in transfected cells.

Chinese hamster ovary cells and NIH 3T3 cells overexpressing mutant human insulin receptors were examined for the presence of hybrid receptors composed of human and rodent insulin receptors. In the present studies, most of the endogenous rodent receptors were found to be immunoprecipitated from the transfected cells but not the parental cells with a monoclonal antibody specific for human receptor. These data indicate that in these transfected cells, most of the endogenous rodent receptors are in a hybrid complex with the overexpressed human receptor. These results together with the in vitro studies of Treadway et al. (Treadway, J.L., Morrison, B.D., Soos, M.A., Siddle, K., Olefsky, J., Ullrich, A., McClain, D.A., and Pessin, J.E. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 214-218) showing that hybrid receptors exhibit transdominant inhibition explain the prior finding indicating that overexpression of defective insulin receptors interferes with the normal signaling of endogenous receptors.     

Definition of IgG- and albumin-binding regions of streptococcal protein G

Protein G, the immunoglobin G-binding surface protein of group C and G streptococci, also binds serum albumin. The albumin-binding site on protein G is distinct from the immunoglobulin G-binding site. By mild acid hydrolysis of the papain-liberated protein G fragment (35 kDa), a 28-kDa fragment was produced which retained full immunoglobulin G-binding activity (determined by Scatchard plotting) but had lost all albumin-binding capacity. A protein G (65 kDa), isolated after cloning and expression of the protein G gene in Escherichia coli, had comparable affinity to immunoglobulin G (5-10 X 10(10)M-1), but much higher affinity to albumin than the 35- and 28-kDa protein G fragments (31, 2.6, and 0 X 10(9)M-1, respectively). The amino-terminal amino acid sequences of the 65-, 35-, and 28-kDa fragments allowed us to exactly locate the three fragments in an overall sequence map of protein G, based on the partial gene sequences published by Guss et al. (Guss, B., Eliasson, M., Olsson, A., Uhlen, M., Frej, A.-K., J?rnvall, H., Flock, J.-I., and Lindberg, M. (1986) EMBO J. 5, 1567-1575) and Fahnestock et al. (Fahnestock, S. R., Alexander, P., Nagle, J., and Filpula, D. (1986) J. Bacteriol. 167, 870-880). In this map could then be deduced the location of three homologous albumin-binding regions and three homologous immunoglobulin G-binding regions.     

Malaria and ovalocytosis--molecular mimicry?

Two recently published reports have described findings which will have a profound impact on the understanding of molecular mechanisms of human resistance to malaria infection. In Melanesian ovalocytosis, a genetic polymorphism found in Papua New Guinea and parts of South East Asia, the red cells are highly resistant to invasion by various species of malaria parasite. The molecular nature of the defect in ovalocytic erythrocytes was not known. Recent reports by Liu et al. (Liu, S.-C., Zhai, S., Palek, J., Golan, D., Amato, D., Hassan, K., Nurse, G., Babona, D., Coetzer, T., Jarolim, P. Zaik, M. and Borwein, S. (1990) N. Engl. J. Med. 323, 1530-1538.) and Jones et al. (Jones, G.L., Edmundson, H.M., Wesche, D. and Saul, A. (1991) Biochim. Biophys. Acta 1096, 33-40.) have now identified the abnormality in the band 3 protein of ovalocytic red cell membranes. A major discovery in the Jones et al. study is the presence of an extended peptide at the N-terminus of ovalocyte band 3 protein. This novel 13 amino acid extended sequence is not found in the primary structure of normal band 3 protein and was suggested to be the cause of band 3 defect in ovalocytes. We have analyzed this extended sequence through Genbank using SWISS-PROT database and found that an almost identical sequence exists in a malaria parasite protein called RESA.     

Prediction of q-values and conformations of gadolinium chelates for magnetic resonance imaging

For gadolinium chelates, we determined that there is a linear correlation between calculated solvent-accessible surface area and q-value, the number of rapidly exchanging water molecules directly bound to the gadolinium ion. A calibration curve was developed to predict q-value based on the solvent-accessible surface area of gadolinium. This predictive method was validated with the following gadolinium crystal structures: (ethylenediaminetetraacetic acid)-gadolinium(III) [Gd(EDTA)] [Templeton, L. K., Templeton, D. H., Zalkin, A., and Ruben, H. W. (1982) Anomalous Scattering by Praseodymium, Samarium, and Gadolinium and Structures of their Thylenediaminetetraacetate (EDTA) Salts. Acta Crystallogr., Sect. B 38, 2155], (1,4,7,10-tetraazacyclododecane-N,N',N' ',N' "-tetraacetic acid)-gadolinium(III) [Gd(DOTA)] [Dubost, J.-P., Leger, J.-M., Langlois, M.-H., Meyer, D., and Schaefer, M. (1991) Structure of a Magnetic Resonance Imaging Agent - The Gadolinium-DOTA Complex C(16)H(24)N(4)O(8)NaGd, 5H(2)O. C. R. Acad. Sci., Ser. 2 312, 349], (diethylenetriaminepentaacetic acid)-gadolinium(III) [Gd(DTPA)] [Stezowski, J. J., and Hoard, J. L. (1984) Heavy Metal Ionophores - Correlations Among Structural Parameters of Complexed Nonpeptide Polyamino Acids. Isr. J. Chem. 24, 323], (diethylenepenta-acetato)-gadolinium(III) [Gd(DTPA-BEA)] [Smith, P. H., Brainard, J. R., Morris, D. E., Jarvinen, G. D., and Ryan, R. R. (1989) Solution and Solid-State Characterization of Europium and Gadolinium Schiff-Base Complexes and Assessment of their Potential as Contrast Agents in Magnetic Resonance Imaging. J. Am. Chem. Soc. 111, 7437], and (1,7,13-triaza-4,10, 16-trioxacyclo-octadecane-N,N',N' '-triacetato)-gadolinium(III) [Gd(TTTA)] [Chen, D., Squattrito, P. J., Martell, A. E., and Clearfield, A. (1990) Synthesis and Crystal Structure of a 9-Coordinate Gadolinium(III) Complex of 1,7,13-Triaza-4,10, 16-Trioxacyclooctadecane-N,N',N' '-Tri-Acetic Acid. Inorg. Chem. 29, 4366]. Predicted q-values were in complete agreement with experimentally determined q-values. A genetic algorithm-based conformational search method was developed to generate valid 3D models for gadolinium chelates. The method was successfully tested on the following gadolinium chelates: Gd(EDTA) (Templeton et al., 1982), Gd(DOTA) (Dubost et al., 1991), Gd(DTPA-BEA) (Smith et al., 1989), Gd(TTTA) (Chen et al., 1990), Gd(triethylene glycol) [Rogers, R. D., Voss, E. J., and Etzenhouser, R. D. (1988) F-Element Crown Ether Complexes. 17. Synthetic and Structural Survey of Lanthanide Chloride Tiethylene Glycol Complexes. Inorg. Chem. 27, 533], and Gd(tetraethylene glycol) [Rogers, R. D., Etzenhouser, R. D., Murdoch, J. S., and Reyes, E. (1991) Macrocycle Complexation Chemistry. 35. Survey of the Complexation of the Open-Chain 15-Crown-5 Analogue tetraethylene Glycol with the Lanthanide Chlorides. Inorg. Chem. 30, 1445].     

Malaria and ovalocytosis — molecular mimicry?

Two recently published reports have described findings which will have a profound impact on the understanding of molecular mechanisms of human resistance to malaria infection. In Melanesian ovalocytosis, a genetic polymorphism found in Papua New Guinea and parts of South East Asia, the red cells are highly resistant to invasion by various species of malaria parasite. The molecular nature of the defect in ovalocytic erythrocytes was not known. Recent reports by Liu et al., (Liu, S.-C., Zhai, S., Palek, J., Golan, D., Amato, D., Hassan, K., Nurse, G., Babona, D., Coetzer, T., Jarolim, P. Zaik, M. and Borwein, S. (1990) N. Engl. J. Med. 323, 1530–1538.) and Jones et al. (Jones, G.L., Edmundson, H.M., Wesche, D. and Saul, A. (1991) Biochim. Biophys. Acta 1096, 33–40.) have now identified the abnormality in the band 3 protein of ovalocytic red cell membranes. A major discovery in the Jones et al, study is the presence of an extended peptide at the N-terminus of ovalocyte band 3 protein. This novel 13 amino acid extended sequence is not found in the primary structure of normal band 3 protein and was suggested to be the cause of band 3 defect in ovalocytes. We have analyzed this extended sequence through Genbank using SWISS-PROT database and found that an almost identical sequence exists in a malaria parasite protein called RESA.     

Receptor activity-modifying protein 1 determines the species selectivity of non-peptide CGRP receptor antagonists

The heterodimeric CGRP receptor requires co-expression of calcitonin receptor-like receptor (CRLR) and an accessory protein called receptor activity-modifying protein (RAMP) 1 (McLatchie, L. M., Fraser, N. J., Main, M. J., Wise, A., Brown, J., Thompson, N., Solari, R., Lee, M. G., and Foord, S. M. (1998) Nature 393, 333-339). Several non-peptide CGRP receptor antagonists have been shown to exhibit marked species selectivity, with >100-fold higher affinities for the human CGRP receptor than for receptors from other species (Doods, H., Hallermayer, G., Wu, D., Entzeroth, M., Rudolf, K., Engel, W., and Eberlein, W. (2000) Br. J. Pharmacol. 129, 420-423; Edvinsson, L., Sams, A., Jansen-Olesen, I., Tajti, J., Kane, S. A., Rutledge, R. Z., Koblan, K. S., Hill, R. G., and Longmore, J. (2001) Eur. J. Pharmacol. 415, 39-44). This observation provided an opportunity to map the determinants of receptor affinity exhibited by BIBN4096BS and the truncated analogs, Compounds 1 and 2. All three compounds exhibited higher affinity for the human receptor, human CRLR/human RAMP1, than for the rat receptor, rat CRLR/rat RAMP1. We have now demonstrated that this species selectivity was directed exclusively by RAMP1. By generating recombinant human/rat CRLR/RAMP1 receptors, we demonstrated that co-expression of human CRLR with rat RAMP1 produced rat receptor pharmacology, and vice versa. Moreover, with rat/human RAMP1 chimeras and site-directed mutants, we have identified a single amino acid at position 74 of RAMP1 that modulates the affinity of small molecule antagonists for CRLR/RAMP1. Replacement of lysine 74 in rat RAMP1 with tryptophan (the homologous amino acid in the human receptor) resulted in a > or =100-fold increase in antagonist affinities, similar to the K(i) values for the human receptor. These observations suggest that important determinants of small molecule antagonist affinity for the CGRP receptor reside within the extracellular region of RAMP1 and provide evidence that this receptor accessory protein may participate in antagonist binding.     

Mutations in the env gene of friend spleen focus-forming virus overcome Fv-2r-mediated resistance to Friend virus-induced erythroleukemia.

Although Fv-2r homozygous mice are resistant to leukemias induced either by an erythropoietin-encoding virus or by wild-type Friend virus (FV) (M. E. Hoatlin, S. L. Kozak, F. Lilly, A. Chakraborti, C. A. Kozak, and D. Kabat, Proc. Natl. Acad. Sci. USA 87:9985-9989, 1990), they are susceptible to some variants of FV (R. A. Steeves, E. A. Mirand, A. Bulba, and P. J. Trudel, Int. J. Cancer 5:349-356, 1970; R. W. Geib, M. B. Seaward, M. L. Stevens, C.-L. Cho, and M. Majumdar, Virus Res. 14:161-174, 1989). To localize the virus gene involved in influencing the host range, we cloned and sequenced the env gene of the BB6 variant of FV (Steeves et al., Int. J. Cancer 5:349-356, 1970). In comparison with the wild-type env gene, the BB6 variant contains a 159-bp deletion that eliminates the membrane-proximal portion of the extracellular domain and 58 point mutations resulting in 13 amino acid changes. Substitution of the variant env gene for the wild-type env gene resulted in a recombinant virus that produced a Friend virus-like disease in Fv-2r homozygotes. Our results identify the spleen focus-forming virus env gene as the viral gene involved in this virus-host interaction. Additionally, they suggest that the product of the Fv-2r gene modifies the interaction between the spleen focus-forming virus envelope protein and the erythropoietin receptor.     

Does correlation of cellulase gene expression and cellulolytic activity in the gut of termite suggest synergistic collaboration of cellulases?

Termites play an important role in degradation of dead plant materials in nature. Over the last century, many researchers have investigated the mechanisms of their lignocellulose digesting system. A recent publication by Zhou et al. (Zhou, X., Smith, J.A., Oi, F.M., Koehler, P.G., Bennett, G.W., Scharf, M.E., 2007. Correlation of cellulase gene expression and cellulolytic activity throughout the gut of the termite Reticulitermes flavipes. Gene 395, 29-39) dealt with the cellulolytic system of the flagellate-harboring termite R. flavipes and suggested "the presence of a single unified cellulose digestion system" in the termite, as an alternative hypothesis of a "dual (i.e. endogenous and symbiotic) cellulose digesting system" proposed by Nakashima et al. (Nakashima, K., Watanabe, H., Saitoh, H., Tokuda, G., Azuma, J.-I., 2002. Dual cellulose-digesting system of the wood-feeding termite, Coptotermes formosanus Shiraki. Insect Biochem. Mol. Biol. 32, 777-784). Here we show that their results actually support a dual cellulose digesting system rather than "a single unified cellulose digestion system". In addition, potential problems with their results are highlighted.     

Coupling of a cloned rat dopamine-D2 receptor to inhibition of adenylyl cyclase and prolactin secretion.

We have previously described a cDNA which encodes a binding site with the pharmacology of the D2-dopamine receptor (Bunzow, J. R., VanTol, H. H. M., Grandy, D. K., Albert, P., Salon, J., Christie, M., Machida, C., Neve, K. A., and Civelli, O. (1988) Nature 336, 783-787). We demonstrate here that this protein is a functional receptor, i.e. it couples to G-proteins to inhibit cAMP generation and hormone secretion. The cDNA was expressed in GH4C1 cells, a rat somatomammotrophic cell strain which lacks dopamine receptors. Stable transfectants were isolated and one clone, GH4ZR7, which had the highest levels of D2-dopamine receptor mRNA on Northern blot, was studied in detail. Binding of D2-dopamine antagonist [3H]spiperone to membranes isolated from GH4ZR7 cells was saturable, with KD = 96 pM, and Bmax = 2300 fmol/mg protein. Addition of GTP/NaCl increased the IC50 value for dopamine competition for [3H]spiperone binding by 2-fold, indicating that the D2-dopamine receptor interacts with one or more G-proteins. To assess the function of the dopamine-binding site, acute biological actions of dopamine were characterized in GH4ZR7 cells. Dopamine, at concentrations found in vivo, decreased resting intra- and extracellular cAMP levels (EC50 = 8 +/- 2 nM) by 50-70% and blocked completely vasoactive intestinal peptide (VIP) induced enhancement of cAMP levels (EC50 = 6 +/- 1 nM). Antagonism of dopamine-induced inhibition of VIP-enhanced cAMP levels by spiperone, (+)-butaclamol, (-)-sulpiride, and SCH23390 occurred at concentrations expected from KI values for these antagonists at the D2-receptor and was stereoselective. Dopamine (as well as several D2-selective agonists) inhibited forskolin-stimulated adenylate cyclase activity by 45 +/- 6%, with EC50 of 500-800 nM in GH4ZR7 membranes. Dopaminergic inhibition of cellular cAMP levels and of adenylyl cyclase activity in membrane preparations was abolished by pretreatment with pertussis toxin (50 ng/ml, 16 h). Dopamine (200 nM) abolished VIP- and thyrotropin-releasing hormone-induced acute prolactin release. These data show conclusively that the cDNA clone encodes a functional dopamine-D2 receptor which couples to G-proteins to inhibit adenylyl cyclase and both cAMP-dependent and cAMP-independent hormone secretion.