Annexin 2 binds and aggregates biological membranes in a Ca(2+)-dependent manner. This protein exists as a monomer (p36) or as a heterotetramer (p90) in which two p36 chains are associated with a dimer of p11, a member of the S100 protein family. Protein kinase C phosphorylates the protein at the level of the N-terminal tail on serines 11 and 25, thereby modifying its oligomeric structure and its properties of membrane aggregation. To analyze these effects, the properties of a series of mutants in which serines 11 and 25 were replaced by alanine and/or glutamic acid were investigated. The affinity for p11 light chain was decreased in the S11E mutants. Glutamic acid residues in positions 11 or 25 did not change membrane binding, either in the tetrameric or in the monomeric form. On the other hand, these mutations affected the aggregation properties of the two forms. For the tetramer, the aggregation efficiency was decreased but not the Ca(2+) sensitivity, whereas the latter was affected in the case of the monomer. The effects were stronger in the S11E mutants, and they were cumulative in the double mutant. They suggest a different conformation of the N-terminal domain in the mutants (and in the phosphorylated protein), a hypothesis which is supported by proteolysis experiments. This conformational change would affect aggregation by the monomer through a dimerization step.

Epinephrine stimulation of rat alpha 2D, alpha 2B, and alpha 2C adrenergic receptor subtypes, expressed stably in Chinese hamster ovary (CHO) cells, caused a rapid, transient activation of mitogen-activated protein kinase (MAPK), with subtype-specific different efficiencies. The order of activation was CHO-2B approximately CHO-2D much greater than CHO-2C. Pertussis toxin blocked the stimulation of MAPK enzymatic activity and the parallel MAPK phosphorylation, demonstrating that these responses are mediated by pertussis toxin-sensitive Gi proteins. Contrary to what has been reported for the alpha 2A subtype expressed in rat-1 fibroblasts, epinephrine did not cause any detectable activation of p21ras in the CHO transfectants. Furthermore, combined application of epinephrine and phorbol myristate acetate had a potent cooperative but not additive effect in clones CHO-2D and CHO-2B but not in CHO-2C, suggesting that protein kinase C is probably differently involved in the signaling by the three alpha 2 receptor subtypes. These results show that in CHO cells, the different alpha 2 adrenergic receptor subtypes utilize differential pathways to activate MAPK in a p21ras-independent way.

This study was designed to determine whether the decrease in serum transthyretin that occurs during food restriction results from gross energy reduction or from depressed protein or lipid intake and to examine the relationship between serum transthyretin and hepatic transthyretin mRNA during moderate protein or food deficiency. Groups of young rats were allowed free access to either a 18% (control) or a 6% protein diet (protein-restricted), or reduced intakes. The food-restricted groups received 60% of control intake from the control diet, a 40% protein-enriched diet, or a 40% lipid-enriched diet, for 28 d. Serum transthyretin concentrations were lower in all experimental groups on d 7 relative to the control group. Control values were reached only in the protein-restricted group by d 14. The low serum transthyretin levels, which were similar in the food-restricted groups, likely resulted from gross energy restriction. Hepatic transthyretin mRNA levels were determined in the control, protein-restricted and food-restricted groups. They were unchanged relative to controls in the protein-restricted group but declined moderately in the food-restricted group on d 7 and 14, before returning to control values by d 28. Thus, the changes in liver transthyretin mRNA levels could partially explain the changes in serum transthyretin in food-restricted rats.

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