Alpha2-adrenoceptor agonists stimulate high-affinity GTPase activity in a receptor subtype-selective manner.

Transfected Chinese hamster ovary cells expressing human alpha2A-, alpha2B- and alpha2C-adrenoceptor subtypes were used to monitor alpha2-adrenoceptor-stimulated GTP hydrolysis. Incubation with 100 microM (-)-adrenaline resulted in stimulation of pertussis toxin-sensitive GTPase by 380% after activation of the alpha2A-subtype, by 320% after activation of the alpha2B-subtype and by 110% after activation of the alpha2C-subtype. The agonists dexmedetomidine, UK14,304 (5-bromo-6-[2-imidazoline-2-ylamino]quinoxaline) and oxymetazoline showed subtype-dependent efficacy. Dexmedetomidine was a full agonist at the alpha2B-subtype and a partial agonist at the alpha2A- and the alpha2C-subtypes. UK14,304 was a full agonist at the alpha2A-subtype and a partial agonist at the other two. Oxymetazoline showed strong partial agonism at the alpha2B-subtype (63% of adrenaline), but did not significantly activate the alpha2A- and the alpha2C-subtypes. These results agreed with cAMP accumulation experiments carried out with cell lines endogenously expressing the alpha2A-subtype (human erythroleukemia, HEL) or the alpha2B-subtype (neuroblastoma-glioma, NG108-15). The GTPase assay may thus provide a valuable tool for the identification of subtype-selective alpha2-adrenoceptor agonists.

Alpha2-adrenoceptor regulation of adenylyl cyclase in CHO cells: dependence on receptor density, receptor subtype and current activity of adenylyl cyclase.

Chinese hamster ovary (CHO) cells stably transfected to express different densities of the human alpha2A-, alpha2B- and alpha2C-adrenoceptor subtypes, were used to characterize the regulation of adenylyl cyclase activity by alpha2-adrenoceptor agonists. In isolated cell membranes, activation of alpha2A- and alpha2C-adrenoceptors did not affect basal enzyme activity, but activation of alpha2B-adrenoceptors stimulated adenylyl cyclase activity. The extent of stimulation was dependent on the receptor density and was insensitive to pertussis toxin treatment. In the presence of 10 microM forskolin all three receptor subtypes mediated inhibition of adenylyl cyclase activity in a pertussis toxin-sensitive manner. In experiments performed with intact cells the same pattern could be seen: the basal production of cAMP was not affected when alpha2C-adrenoceptors were activated, but activated alpha2B-adrenoceptors mediated stimulation of cAMP production. In the presence of forskolin, both receptor subtypes mediated inhibition of cAMP production. Our results suggest that alpha2B-adrenoceptors are coupled to both Gi and Gs proteins. The signal transduction pathway to which the receptor is coupled is not dependent on receptor density, but its effect on adenylyl cyclase regulation is dependent on the current activity of adenylyl cyclase. The results also suggest that the alpha2A- and alpha2C-subtypes are preferentially coupled to Gi and transduce only inhibition of adenylyl cyclase activity in transfected CHO cells. At low densities of alpha2C-adrenoceptors, clonidine was a partial agonist, but in clones expressing high levels of alpha2C-adrenoceptors, clonidine acted as a full agonist by inhibiting cAMP accumulation with the same efficacy as (-)-noradrenaline. This demonstrates that receptor reserve can mask partial agonist activity.

Similar ligand binding in recombinant human alpha 2 C2-adrenoceptors produced in mammalian, insect and yeast cells.

Ligand binding properties were investigated in recombinant human alpha 2C2-adrenoceptors expressed in three different host systems: Shionogi S115 mouse mammary tumour cells, Spodoptera frugiperda Sf9 insect cells and Saccharomyces cerevisiae yeast cells. The expected 43 kDa alpha 2C2 protein was visualized with immunoblotting using a polyclonal alpha 2C2-receptor antibody. [3H]Rauwolscine binding in cell homogenates or membranes (Bmax 3-11 pmol/mg protein; Kd approximately 5.5 nM) was inhibited by prazosin, oxymetazoline, RX821002, chlorpromazine and (-)-noradrenaline with and without the GTP-analogue Gpp(NH)p with similar Ki values in the different host systems. This indicates that alpha 2C2-adrenoceptors retain their binding characteristics irrespective of the host environment.

Genetic engineering of Escherichia coli inorganic pyrophosphatase. Tyr55 and Tyr141 are important for the structural integrity.

Two tyrosines are supposed to be essential for the activity and to participate in the stabilization of Escherichia coli inorganic pyrophosphatase (PPiase) against heat denaturation [Samejima, T., Tamagawa, Y., Kondo, Y., Hachimori, A., Kaji, H., Takeda, A. and Shiroya, Y. (1988) J. Biochem. (Tokyo) 103, 766-772]. To locate these two tyrosines in the amino acid sequence, we substituted all the eight tyrosines of E. coli PPiase with phenylalanine and studied the properties of these YF mutant PPiases. Interestingly, substitution of the tyrosines (Tyr51, Tyr55 and Tyr141) conserved with the amino acid sequence of yeast PPiase [Lahti, R., Kolakowski, L. F., Heinonen, J., Vihinen, M., Pohjanoksa, K. and Cooperman, B. (1990) Biochim. Biophys. Acta 1038, 338-345] exerted the most drastic effects on the structure and activity of E. coli PPiase. PPiase variants YF51, YF55 and YF141 had 64%, 7% and 22% of the wild-type PPiase activity, respectively. Furthermore, PPiase variant YF141 had an increased sensitivity to heat denaturation, whereas mutant PPiase YF55 displayed a profound conformational change, as demonstrated by the binding of the fluorescent dye 9-(diethylamino)-5H-benzo(alpha) phenoxazine-5-one (Nile red) that monitors the hydrophobicity of protein surfaces. None of the tyrosines of E. coli PPiase seem to be essential for catalysis, but Tyr55 and Tyr141 are important for the structural integrity of E. coli PPiase.

A site-directed mutagenesis study on Escherichia coli inorganic pyrophosphatase. Glutamic acid-98 and lysine-104 are important for structural integrity, whereas aspartic acids-97 and -102 are essential for catalytic activity.

Analysis of the conservation of functional residues between yeast and Escherichia coli inorganic pyrophosphatases (PPases) suggested that Asp-97, Glu-98, Asp-102, and Lys-104 are important for the action of E. coli PPase [Lahti, R., Kolakowski, L. F., Heinonen, J., Vihinen, M., Pohjanoksa, K., & Cooperman, B. S. (1990) Biochim. Biophys. Acta 1038, 338-345]. We replaced these four residues by oligonucleotide-directed mutagenesis, giving variant PPases DV97, DE97, EV98, DV102, DE102, KI104, and KR104. PPase variants DV97, DV102, and KI104 had no enzyme activity, whereas PPase variants DE97, EV98, DE102, and KR104 had 22%, 33%, 3%, and 3% of the wild-type PPase activity, respectively. This suggests that Asp-97, Asp-102, and Lys-104 are essential for the catalytic activity of E. coli PPase. PPase variants DV98 and KR104 also had an increased sensitivity to heat denaturation; incubation of these mutant PPases at 75 degrees C for 15 min in the presence of 5 mM magnesium ion decreased the activity to 20% and 1%, respectively, of the initial value while 74% of the activity was observed with wild-type PPase. Furthermore, these thermolabile mutant PPases displayed the most profound conformational changes of the PPase variants examined, as demonstrated by the binding of the fluorescent dye Nile red that monitors the hydrophobicity of protein surfaces. Accordingly, Glu-98 and Lys-104 seem to be important for the structural integrity of E. coli PPase.

Conservation of functional residues between yeast and E. coli inorganic pyrophosphatases.

The alignments of the amino acid sequences of inorganic pyrophosphatase (PPase) from Saccharomyces cerevisiae (Y1-PPase, 286 amino acids) and Escherichia coli (E-PPase, 175 amino acids) are examined in the light of crystallographic and chemical modification results placing specific amino acid residues at the active site of the yeast enzyme. The major results are: (1) the full E-PPase sequence aligns within residues 28-225 of Y1-PPase, raising the possibility that the N-terminal and C-terminal portions of Y1-PPase may not be essential for activity, and (2) that whereas the overall identity between the two sequences is only modest (22-27% depending on the choice of alignment parameters), of some 17 putative active site residues, 14-16 are identical between Y-PPase and E-PPase. PPase thus appears to be an example of enzymes from widely divergent species that conserve common functional elements within the context of substantial overall sequence variation.