Cyclic 3′‐5′‐adenosine monophosphate binds to annexin I and regulates calcium‐dependent membrane aggregation and ion channel activity

Abstract

The annexin (Anx) gene family comprises a set of calcium-dependent membrane binding proteins, which have been implicated in a wide variety of cellular processes including membrane fusion and calcium channel activity. We report here that cAMP activates CaZ+-dependent aggregation of both phosphatidylserine (PS) liposomes and bovine chromaffin granules driven by ides 1-12]annexin 1 (lipocortin I, Anxl). The mechanism of cAMP action involves an increase in Anxl-dependent cooperativity on the rate of such a reaction without affecting the corresponding k,2 values. Cyclic AMP causes the values of the Hill coefficient (nil) for AnxI to change from 3 to 6 in both PS liposomes and chromaffin granules. By contrast, ATP inhibits the rate of aggregation activity without affecting the cooperativity or the extent of aggregation process. We were also able to photolabel Anxl specifically with an 8-azido analogue of cAMP by a calcium-independent process. Such a process is saturable, yielding a K d = 0.8 pM by Scatchard analysis. Specific displacement occurs in the presence of cAMP and ATP. Finally, we found that cAMP alters the conductance of calcium channels formed by Anxl in planar lipid bilayers. We interpret these data to indicate that Anxl binds both calcium and cAMP independently, and that both actions have functional consequences. This is the first report of a nucleotide binding function for a member of the annexin gene family.

Annexins as nucleotide‐binding proteins: Facts and speculations

“…They exhibit some homology to cAMP-binding proteins but the significance of these similarities is not yet apparent. (30) The binding properties of annexins also point to differences between various tissueand specimen-specific isoforms of annexins with respect to their ability to interact with nucleotides. (30,32,34) The ATPdependent processes in which annexins may be implicated are summarized in Fig.…”

“…(31) ATP inclusion in the trans chamber affects the ion channel activity of D-AnxI incorporated into the planar lipid bilayer. (30) Moreover, ATP (1 mM) inhibits the aggregation of chromaffin granules and PS liposomes induced by AnxI in the presence of Ca 2 . (30) AnxI also exhibited binding properties for cAMP, but not GTP or cGMP.…”

“…(30) Moreover, ATP (1 mM) inhibits the aggregation of chromaffin granules and PS liposomes induced by AnxI in the presence of Ca 2 . (30) AnxI also exhibited binding properties for cAMP, but not GTP or cGMP. (30) Nucleotide-dependent mechanisms are also observed with other annexins: (1) binding of AnxVI to the hepatocyte plasma membrane is stimulated in the presence of physiological concentrations of ATP, however, in relatively high concentrations of Ca 2 , (58) (2) Ca 2 -dependent binding of AnxVI to model membranes (erythrocyte ghosts) in the presence of ATP is accompanied by a one order of magnitude shift to higher values of Ca 2 concentrations required for halfmaximal stimulation of the process, (32) and (3) Ca 2dependent aggregation of PS liposomes evoked by AnxVI is stimulated by ATP at low, submicromolar Ca 2 concentrations, but is inhibited by higher, non-physiological concentrations of Ca 2 .…”

“…(30) AnxI also exhibited binding properties for cAMP, but not GTP or cGMP. (30) Nucleotide-dependent mechanisms are also observed with other annexins: (1) binding of AnxVI to the hepatocyte plasma membrane is stimulated in the presence of physiological concentrations of ATP, however, in relatively high concentrations of Ca 2 , (58) (2) Ca 2 -dependent binding of AnxVI to model membranes (erythrocyte ghosts) in the presence of ATP is accompanied by a one order of magnitude shift to higher values of Ca 2 concentrations required for halfmaximal stimulation of the process, (32) and (3) Ca 2dependent aggregation of PS liposomes evoked by AnxVI is stimulated by ATP at low, submicromolar Ca 2 concentrations, but is inhibited by higher, non-physiological concentrations of Ca 2 . (43) Several annexins exhibit various affinities for purine nucleotides, as demonstrated by the covalent labeling of annexins with nucleotide derivatives (azido-ATP), (30,32± 34) as well as by circular dichroism, (42) Fourier transform infrared spectroscopy (42) and fluorescence spectroscopy.…”

“…(32,43) Comparison of two homologous members of the annexin family, isolated from the same animal specimen and the same tissue, AnxIV and AnxVI, which differ in their number of Ca 2 -and phospholipid-binding sites, revealed differences in nucleotide-binding properties. (32) In addition, it was found that AnxI may bind cAMP (30) while AnxVII is probably a GTPase. (34,35) GTP modulates the pH-dependent, Ca 2 -specific channel activity of AnxV (44) and plant annexins were found to catalyze the hydrolysis of ATP/GTP.…”