Allen R. Rhoads

Calmodulin signaling via the IQ motif.

The IQ motif is widely distributed in both myosins and non‐myosins and is quite common in the database that includes more than 900 Pfam entries. An examination of IQ motif‐containing proteins that are known to bind calmodulin (CaM) indicates a wide diversity of biological functions that parallel the Ca2+‐dependent targets. These proteins include a variety of neuronal growth proteins, myosins, voltage‐operated channels, phosphatases, Ras exchange proteins, sperm surface proteins, a Ras Gap‐like protein, spindle‐associated proteins and several proteins in plants. The IQ motif occurs in some proteins with Ca2+‐dependent CaM interaction where it may promote Ca2+‐independent retention of CaM. The action of the IQ motif may result in complex signaling as observed for myosins and the L‐type Ca2+ channels and is highly localized as required for sites of neuronal polarized growth and plasticity, fertilization, mitosis and cytoskeletal organization. The IQ motif associated with the unconventional myosins also promotes Ca2+ regulation of the vectorial movement of cellular constituents to these sites. Additional regulatory roles for this versatile motif seem likely.

ATP-induced secretion in PC12 cells and photoaffinity labeling of receptors

Abstract— Secretion of catecholamines by rat PC12 cells is strongly stimulated by extracellular ATP via a P2‐type pur‐inergic receptor. ATP‐induced norepinephrine release was inhibited 80% when extracellular Ca2+ was absent. Only four nucleotides, ATP, ATPγS, benzoylbenzoyl ATP (BzATP), and 2‐methylthio‐ATP, gave substantial stimulation of norepinephrine release from PC12 cells. ATP‐induced secretion was inhibited by Mg2+, and this inhibition was overcome by the addition of excess ATP suggesting that ATP4‐was the active ligand. ATP‐induced secretion of catecholamine release was enhanced by treatment of cells with pertussis toxin or 12‐O‐tetradecanoylphorbol 13‐acetate. The stimulatory effects of 12‐O‐tetradecanoyl‐phorbol 13‐acetate and pertussis toxin on norepinephrine release were additive. After brief exposure of intact cells to the photoaffinity analog, [α‐32P]BzATP, two major proteins of 44 and 50 kDa and a minor protein of 97 kDa were labeled. An excess of ATP‐γS and BzATP but not GTP blocked labeling of the proteins by [32P]BzATP. Labeling of the 50‐kDa protein was more sensitive to competition by 2‐methylthio‐ATP than the other labeled proteins, suggesting that the 50‐kDa protein represents the P2 receptor responsible for ATP‐stimulated secretion in these cells.

Hydrolysis-Resistant GTP Analogs Stimulate Catecholamine Release from Digitonin-Permeabilized PC12 Cells

Abstract: The effect of the hydrolysis‐resistant GTP analogs, guanosine 5′‐O‐(3‐thiotriphosphate) (GTPγS) and guanylyl imidodiphosphate (GMPPNP), on norepinephrine (NE) secretion from digitonin‐permeabilized rat pheochromocytoma cells, PC12, was examined. Although secretion in the presence of saturating Ca2+ (10 μM) was not affected by GTP7S or GMPPNP, secretion in the absence of Ca2+ was stimulated by these GTP analogs. Secretion induced by saturating concentrations of GTPγS or GMPPNP was approximately 80% of that induced by 10 μM Ca2+. Half‐maximum stimulation was induced by 30 μM GTPγS or GMPPNP. Both Ca2+‐stimulated and GTPγS‐stimulated secretion were ATP dependent and inhibited by N‐ethylmaleimide. The GTPγS‐stimulated secretion of NE from permeabilized PC12 cells does not appear to result from either the release of Ca2+ or the activation of protein kinase C. Activation of protein kinase C by pretreatment of intact cells with 12‐O‐tetradecanoyl‐phorbol 13‐acetate caused a 50% increase in both Ca2+‐stimulated and GTP7S‐stimulated secretion. Cholera and pertussis toxins did not affect Ca2+‐stimulated or GTPγS‐stim‐ulated NE secretion. Guanosine 5′‐O‐(2‐thiodiphosphate) (GDPβS) and GTP inhibited GTPγS‐stimulated secretion but not Ca2+‐stimulated secretion. The inability of GDPβS to inhibit Ca2+‐stimulated secretion indicates that the process affected by GTPγS is not an essential step in the Ca2+‐stimulated pathway.

Multiple calmodulin genes in fish

In mammals, identical calmodulin (CaM) proteins are encoded by three nonallelic genes that differ in their promoter regions and untranslated regions (UTRs). The UTRs of each of these three genes are specific for each gene and are highly conserved. In this study, sequences obtained from the GenBank and EST databases and sequencing were examined for several species of fish to ascertain whether this multi-gene one protein system exhibited in mammals extends to other vertebrates. Three genes in zebrafish (Danio rerio) designated α, β, and γwere identified. As in mammals, these genes differ in the 3′-UTR region but encode completely identical CaMs. PCR primers spanning the coding and the 3′-UTR regions were designed based on the assembled sequences and used to confirm the presence of each gene in the cDNA library. Other species of fish were also found to contain homologous genes that were closely related as indicated by phylogenetic analysis. The 3′-UTR of the α, βand particularly the γCaM gene of fish were not found to be as conserved as the corresponding genes of mammalian species possibly due to the span of evolutionary time. Only a few short elements in the 3′-UTR were observed to be similar in fish and mammals. These short regions of identity are shared primarily between the mammalian CaM II and CaM I and the αgene and βgene of fish, respectively. Thus, the multi-gene one protein system occurs among fish as well as among mammals.

Particulate cyclic 3′,5′-nucleotide phosphodiesterase and calmodulin of cardiac muscle

Cyclic AMP and cyclic GMP phosphodiesterase and calmodulin were measured in purified subcellular fractions of cardiac muscle. Phosphodiesterase activity solubilized by sonication of the nuclear fraction yielded a major 6.6 S form which was calcium-sensitive and cyclic GMP-specific. Phosphodiesterase activity occurring in the nuclear fraction could be further enriched by subfractionation on sucrose density gradients in the presence of MgCl2.

Small membrane-associated gtp-binding proteins of catecholamine-secreting cells

1. Four GTP-binding proteins (23-27 kDa) were identified in membranes from PC12 cells by [alpha 32P]GTP binding to nitrocellulose blots of SDS-polyacrylamide gels. 2. The GTP-binding proteins remained associated with membranes during stimulation of intact cells by K(+)-depolarization or even after addition of Ca2+ to digitonin-permeabilized cells. 3. By two-dimensional gel electrophoresis, six GTP-binding proteins were resolved and based on their mobility, their phosphorylation state appeared independent of Ca2+. 4. Fractionation of PC12 membranes showed that these GTP-binding proteins were broadly distributed in post-nuclear membranes with the plasma membranes containing the highest specific GTP-binding activity. 5. Membrane fractions from bovine adrenal medulla contain similar GTP-binding proteins with GTP-binding intensity also being highest in the plasma membrane. 6. The GTP-binding proteins could be concentrated in the detergent-rich fraction upon Triton X-114 phase separation.

Age and detection of retroprocessed pseudogenes in murine rodents.

Abstract. Retroprocessed pseudogenes, calmodulin II (ψ1, ψ2, and ψ3 CALMII), ψα-tubulin, π-glutathione S-transferase (ψπ-GST) from rat, lactic acid dehydrogenase (ψ LDH) from mouse, and heat shock protein 60 chaperonin (ψ HSP60) from Chinese hamster, were examined for their presence in these species by polymerase chain reaction (PCR). Pseudogenes of these murine rodents were detected by PCR only in those species in which the genes were originally identified, suggesting that the selected pseudogene of one species arose too recently to be detected in the genomes of the other rodent species. The calculated ages of the rodent pseudogenes ranged from 1.7 Myr (ψα-tubulin) to 7.5 Myr (ψ3 CALMII) when employing a homologous functional gene of the taxon as a reference in the relative rate test with the mouse or rat as the outgroup. Given the high rate of divergence of the genes of rodents relative to other species, selection of an outgroup with similar mutation rates seems warranted. To justify further the conclusion that the selected pseudogenes were indeed retroprocessed after these three taxa diverged, the presence of the pseudogenes in the genome of different rat species was examined. The existence of ψ3 CALMII and ψα-tubulin pseudogenes of Rattus norvegicus among species belonging to Rattus sensu stricto is evidence for the common ancestry of this group.