Skarlatos G. Dedos

Selective coupling of type 6 adenylyl cyclase with type 2 IP3 receptors mediates direct sensitization of IP3 receptors by cAMP

Interactions between cyclic adenosine monophosphate (cAMP) and Ca2+ are widespread, and for both intracellular messengers, their spatial organization is important. Parathyroid hormone (PTH) stimulates formation of cAMP and sensitizes inositol 1,4,5-trisphosphate receptors (IP3R) to IP3. We show that PTH communicates with IP3R via “cAMP junctions” that allow local delivery of a supramaximal concentration of cAMP to IP3R, directly increasing their sensitivity to IP3. These junctions are robust binary switches that are digitally recruited by increasing concentrations of PTH. Human embryonic kidney cells express several isoforms of adenylyl cyclase (AC) and IP3R, but IP3R2 and AC6 are specifically associated, and inhibition of AC6 or IP3R2 expression by small interfering RNA selectively attenuates potentiation of Ca2+ signals by PTH. We define two modes of cAMP signaling: binary, where cAMP passes directly from AC6 to IP3R2; and analogue, where local gradients of cAMP concentration regulate cAMP effectors more remote from AC. Binary signaling requires localized delivery of cAMP, whereas analogue signaling is more dependent on localized cAMP degradation.

Regulation of Inositol 1,4,5-Trisphosphate Receptors by cAMP Independent of cAMP-dependent Protein Kinase

In HEK cells stably expressing type 1 receptors for parathyroid hormone (PTH), PTH causes a sensitization of inositol 1,4,5-trisphosphate receptors (IP3R) to IP3 that is entirely mediated by cAMP and requires cAMP to pass directly from type 6 adenylyl cyclase (AC6) to IP3R2. Using DT40 cells expressing single subtypes of mammalian IP3R, we demonstrate that high concentrations of cAMP similarly sensitize all IP3R isoforms to IP3 by a mechanism that does not require cAMP-dependent protein kinase (PKA). IP3 binding to IP3R2 is unaffected by cAMP, and sensitization is not mediated by the site through which ATP potentiates responses to IP3. In single channel recordings from excised nuclear patches of cells expressing IP3R2, cAMP alone had no effect, but it increased the open probability of IP3R2 activated by a submaximal concentration of IP3 alone or in combination with a maximally effective concentration of ATP. These results establish that cAMP itself increases the sensitivity of all IP3R subtypes to IP3. For IP3R2, this sensitization results from cAMP binding to a novel site that increases the efficacy of IP3. Using stably expressed short hairpin RNA to reduce expression of the G-protein, Gαs, we demonstrate that attenuation of AC activity by loss of Gαs more substantially reduces sensitization of IP3R by PTH than does comparable direct inhibition of AC. This suggests that Gαs may also specifically associate with each AC·IP3R complex. We conclude that all three subtypes of IP3R are regulated by cAMP independent of PKA. In HEK cells, where IP3R2 selectively associates with AC6, Gαs also associates with the AC·IP3R signaling junction.

IP3 receptors: some lessons from DT40 cells.

Summary:  Inositol‐1,4,5‐trisphosphate receptors (IP3Rs) are intracellular Ca2+ channels that are regulated by IP3 and Ca2+ and are modulated by many additional signals. These properties allow them to initiate and, via Ca2+‐induced Ca2+ release, regeneratively propagate Ca2+ signals evoked by receptors that stimulate formation of IP3. The ubiquitous expression of IP3R highlights their importance, but it also presents problems when attempting to resolve the behavior of defined IP3R. DT40 cells are a pre‐B‐lymphocyte cell line in which high rates of homologous recombination afford unrivalled opportunities to disrupt endogenous genes. DT40‐knockout cells with both alleles of each of the three IP3R genes disrupted provide the only null‐background for analysis of homogenous recombinant IP3R. We review the properties of DT40 cells and consider three areas where they have contributed to understanding IP3R behavior. Patch‐clamp recording from the nuclear envelope and Ca2+ release from intracellular stores loaded with a low‐affinity Ca2+ indicator address the mechanisms leading to activation of IP3R. We show that IP3 causes intracellular IP3R to cluster and re‐tune their responses to IP3 and Ca2+, better equipping them to mediate regenerative Ca2+ signals. Finally, we show that DT40 cells reliably count very few IP3R into the plasma membrane, where they mediate about half the Ca2+ entry evoked by the B‐cell antigen receptor.

Ca2+ as second messenger in PTTH-stimulated prothoracic glands of the silkworm, Bombyx mori

Abstract Measurements of Ca 2+ influx and [Ca 2+ ] i changes in Fura-2/AM-loaded prothoracic glands (PGs) of the silkworm, Bombyx mori , were used to identify Ca 2+ as the actual second messenger of the prothoracicotropic hormone (PTTH) of this insect. Dose-dependent increases of [Ca 2+ ] i in PG cells were recorded in the presence of recombinant PTTH (rPTTH) within 5 minutes. The rPTTH-mediated increases of [Ca 2+ ] i levels were dependent on extracellular Ca 2+ . They were not blocked by the dihydropyridine derivative, nitrendipine, an antagonist of high-voltage-activated (HVA) Ca 2+ channels, and by bepridil, an antagonist of low-voltage-activated (LVA) Ca 2+ channels. The trivalent cation La 3+ , a non-specific blocker of plasma membrane Ca 2+ channels, eliminated the rPTTH-stimulated increase of [Ca 2+ ] i levels in PG cells and so did amiloride, an inhibitor of T-type Ca 2+ channels. Incubation of PG cells with thapsigargin resulted in an increase of [Ca 2+ ] i levels, which was also dependent on extracellular Ca 2+ and was quenched by amiloride, suggesting the existence of store-operated plasma membrane Ca 2+ channels, which can also be inhibited by amiloride. Thapsigargin and rPTTH did not operate independently in stimulating increases of [Ca 2+ ] i levels and one agent’s mediated increase of [Ca 2+ ] i was eliminated in the presence of the other. TMB-8, an inhibitor of intracellular Ca 2+ release from inositol 1,4,5 trisphosphate (IP 3 )-sensitive Ca 2+ stores, blocked the rPTTH-stimulated increases of [Ca 2+ ] i levels, suggesting an involvement of IP 3 in the initiation of the rPTTH signaling cascade, whereas ryanodine did not influence the rPTTH-stimulated increases of [Ca 2+ ] i levels. The combined results indicate the presence of a cross-talk mechanism between the [Ca 2+ ] i levels, filling state of IP 3 -sensitive intracellular Ca 2+ stores and the PTTH-receptor’s-mediated Ca 2+ influx.

Counting Functional Inositol 1,4,5-Trisphosphate Receptors into the Plasma Membrane

Inositol 1,4,5-trisphosphate receptors (IP3R) within the endoplasmic reticulum mediate release of Ca2+ from intracellular stores. Different channels usually mediate Ca2+ entry across the plasma membrane. In B lymphocytes and a cell line derived from them (DT40 cells), very few functional IP3R(∼2/cell) are invariably expressed in the plasma membrane, where they mediate about half the Ca2+ entry evoked by activation of the B-cell receptor. We show that cells reliably count ∼2 functional IP3R into the plasma membrane even when their conductance and ability to bind IP3 are massively attenuated. We conclude that very small numbers of functional IP3R can be reliably counted into a specific membrane compartment in the absence of feedback signals from the active protein.

Adenophostin A and analogues modified at the adenine moiety: synthesis, conformational analysis and biological activity

The synthesis of adenophostin A (2) and two analogues [etheno adenophostin (4) and 8-bromo adenophostin (5)] modified at the adenine moiety, is reported. A combination of NMR analysis and molecular modelling was used to compare their structures in solution and determined that they all adopt very similar conformations. The analogues were tested for their ability to mobilise Ca(2+) from DT40 cells expressing recombinant Type 1 rat Ins(1,4,5)P(3)R which reveals etheno adenophostin as a high affinity fluorescent probe of the Ins(1,4,5)P(3)R. 8-Bromo adenophostin was only slightly less potent. The biological results support our current hypothesis regarding the binding mode of adenophostin A at the Ins(1,4,5)P(3)R, i. e. that a cation-pi interaction between the base moiety and Arg 504 of the receptor in combination with H-bonding may be responsible for the high potency of adenophostin A relative to Ins(1,4,5)P(3).

Activation of IP3 receptors by synthetic bisphosphate ligands

Ca(2+) release by d-myo-inositol 1,4,5-trisphosphate receptors (IP(3)Rs) is widely considered to require the vicinal 4,5-bisphosphate motif of IP(3), with P-5 and P-4 engaging the alpha and beta domains of the binding site; using synthesis and mutagenesis we show that the adenine of synthetic glyconucleotides, through an interaction with Arg504, can replace the interaction of either P-1 or P-5 with the alpha-domain producing, respectively, the most potent bisphosphate agonist yet synthesised and the first agonist of IP(3)R without a vicinal bisphosphate motif; this will stimulate new approaches to IP(3)R ligand design.

A missense mutation in Fgfr1 causes ear and skull defects in hush puppy mice

The hush puppy mouse mutant has been shown previously to have skull and outer, middle, and inner ear defects, and an increase in hearing threshold. The fibroblast growth factor receptor 1 (Fgfr1) gene is located in the region of chromosome 8 containing the mutation. Sequencing of the gene in hush puppy heterozygotes revealed a missense mutation in the kinase domain of the protein (W691R). Homozygotes were found to die during development, at approximately embryonic day 8.5, and displayed a phenotype similar to null mutants. Reverse transcription PCR indicated a decrease in Fgfr1 transcript in heterozygotes and homozygotes. Generation of a construct containing the mutation allowed the function of the mutated receptor to be studied. Immunocytochemistry showed that the mutant receptor protein was present at the cell membrane, suggesting normal expression and trafficking. Measurements of changes in intracellular calcium concentration showed that the mutated receptor could not activate the IP3 pathway, in contrast to the wild-type receptor, nor could it initiate activation of the Ras/MAP kinase pathway. Thus, the hush puppy mutation in fibroblast growth factor receptor 1 appears to cause a loss of receptor function. The mutant protein appears to have a dominant negative effect, which could be due to it dimerising with the wild-type protein and inhibiting its activity, thus further reducing the levels of functional protein. A dominant modifier, Mhspy, which reduces the effect of the hush puppy mutation on pinna and stapes development, has been mapped to the distal end of chromosome 7 and may show imprinting.

Signalling from parathyroid hormone.

PTH (parathyroid hormone), acting via type 1 PTH receptors, is a major regulator of plasma [Ca(2+)]. The G-protein, G(s), is an essential component of the sequence linking PTH to plasma Ca(2+) regulation, but the relative importance of intracellular signals, including Ca(2+) and cAMP, that lie downstream of G(s) is not resolved.

Selective coupling of type 6 adenylyl cyclase with type 2 IP3receptors mediates direct sensitization of IP3receptors by cAMP

Tovey et al. 2008. J. Cell Biol. doi:10.1083/jcb.200803172 [OpenUrl][1][Abstract/FREE Full Text][2] [1]: {openurl}?query=rft_id%253Dinfo%253Adoi%252F10.1083%252Fjcb.200803172%26rft_id%253Dinfo%253Apmid%252F18936250%26rft.genre%253Darticle%26rft_val_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%