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PubMed1991

Kinetics and nucleotide specificity of a surface cAMP binding site in Dictyostelium discoideum, which is not down-regulated by cAMP.

Dictyostelium cells exhibit four types of kinetically distinct surface cAMP binding sites, the AH, AL, BS, and BSS sites, which are down-regulated during persistent stimulation with cAMP. Although most cAMP-induced responses are subject to desensitization during constant stimulation, some responses, notably the induction of post-aggregative gene expression, require persistent cAMP stimulation. The kinetics and specificity of residual cAMP-binding activity in cells treated for 4 h with micromolar cAMP were investigated. It was found that around 4000 rapidly dissociating binding sites per cell with an affinity of about 300 nM are retained after down-regulation. The nucleotide specificity of the remaining sites was very similar, but not completely identical to the AH, AL and B sites, suggesting that these sites belong to the same class of cell surface cAMP receptors and may be utilized to mediate responses requiring continuous cAMP stimulation.

Animals↗
PubMed2000

Consequences of cAMP-binding site mutations on the structural stability of the type I regulatory subunit of cAMP-dependent protein kinase.

The regulatory (R) subunit of cAMP-dependent protein kinase (cAPK) is a multidomain protein with two tandem cAMP-binding domains, A and B. The importance of cAMP binding on the stability of the R subunit was probed by intrinsic fluorescence and circular dichroism (CD) in the presence and absence of urea. Several mutants were characterized. The site-specific mutants R(R209K) and R(R333K) had defects in cAMP-binding sites A and B, respectively. R(M329W) had an additional tryptophan in domain B. Delta(260-379)R lacked Trp260 and domain B. The most destabilizing mutation was R209K. Both CD and fluorescence experiments carried out in the presence of urea showed a decrease in cooperativity of the unfolding, which also occurred at lower urea concentrations. Unlike native R, R(R209K) was not stabilized by excess cAMP. Additionally, CD revealed significant alterations in the secondary structure of the R209K mutant. Therefore, Arg209 is important not only as a contact site for cAMP binding but also for the intrinsic structural stability of the full-length protein. Introducing the comparable mutation into domain B, R333K, had a smaller effect on the integrity and stability of domain A. Unfolding was still cooperative; the protein was stabilized by excess cAMP, but the unfolding curve was biphasic. The R(M329W) mutant behaved functionally like the native protein. The Delta(260-379)R deletion mutant was not significantly different from wild-type RIalpha in its stability. Consequently, domain B and the interaction between Trp260 and cAMP bound to site A are not critical requirements for the structural stability of the cAPK regulatory subunit.

Binding Sites↗
PubMed2006

Kinetic and structural studies of the allosteric conformational changes induced by binding of cAMP to the cAMP receptor protein from Escherichia coli.

The cAMP receptor protein, allosterically activated by cAMP, regulates the expression of more than 100 genes in Escherichia coli. CRP is a homodimer of two-domain subunits. It has been suggested that binding of cAMP to CRP leads to a long-distance signal transduction from the N-terminal cAMP binding domain to the C-terminal domain of the protein responsible for interaction with specific sequences of DNA. In this study, the stopped-flow and time-resolved fluorescence lifetime measurements were used to observe the kinetics of the distance changes between the N-terminal and C-terminal domain of CRP induced by binding of cAMP to high-affinity binding sites. In these measurements, we used the constructed CRP heterodimer, which possesses a single Trp85 residue localized at the N-terminal domain of one CRP subunit, and fluorescently labeled by 1,5-I-AEDANS Cys178 localized at the C-terminal domain of the same subunit or at the opposite one. The Förster resonance energy transfer method has been used to study the distance changes, induced by binding of cAMP, between Trp85 (fluorescence donor) and Cys178-AEDANS (fluorescence acceptor) in the CRP structure. The obtained results show that the allosteric transitions of CRP at micromolar cAMP concentrations follow the sequential binding model, in which binding of cAMP to high-affinity sites causes a 4 A movement of the C-terminal domain toward N-terminal domains of the protein, with kinetics faster than 2 ms, and CRP adopts the "closed" conformation. This fast process is followed by the slower reorientation of both CRP subunits.

Allosteric Regulation↗
PubMed2004

Detecting cAMP-induced Epac activation by fluorescence resonance energy transfer: Epac as a novel cAMP indicator.

Epac1 is a guanine nucleotide exchange factor for Rap1 that is activated by direct binding of cAMP. In vitro studies suggest that cAMP relieves the interaction between the regulatory and catalytic domains of Epac. Here, we monitor Epac1 activation in vivo by using a CFP-Epac-YFP fusion construct. When expressed in mammalian cells, CFP-Epac-YFP shows significant fluorescence resonance energy transfer (FRET). FRET rapidly decreases in response to the cAMP-raising agents, whereas it fully recovers after addition of cAMP-lowering agonists. Thus, by undergoing a cAMP-induced conformational change, CFP-Epac-YFP serves as a highly sensitive cAMP indicator in vivo. When compared with a protein kinase A (PKA)-based sensor, Epac-based cAMP probes show an extended dynamic range and a better signal-to-noise ratio; furthermore, as a single polypeptide, CFP-Epac-YFP does not suffer from the technical problems encountered with multisubunit PKA-based sensors. These properties make Epac-based FRET probes the preferred indicators for monitoring cAMP levels in vivo.

Animals↗
PubMed1997

Novel bradykinin signalling events in PC-12 cells: stimulation of the cAMP pathway leads to cAMP-mediated translocation of protein kinase Cepsilon.

In the rat pheochromocytoma cell line PC-12, bradykinin (BK) stimulated phosphatidylinositol hydrolysis by 4-5-fold and, additionally, intracellular cAMP accumulation by approx. 1.6-fold. EC50 values for BK were 3 nM and 2 nM respectively. The BK-induced increase in cAMP accumulation was paralleled by a 1.6-fold increase in protein kinase A (PKA) activity. The time course of BK-stimulated inositol phosphate formation was rapid (t1/2<1 min), whereas the BK-induced cAMP accumulation was lagging (t1/2 approx. 6 min). The effect of BK on the cAMP pathway was independent of pertussis toxin, excluding an indirect stimulation of adenylate cyclase via betagamma-complexes from Gi or Go proteins. Two different protein kinase C (PKC) inhibitors, bisindolylmaleimide and Ro 31-820, failed to prevent BK-induced cAMP accumulation, and exclude PKC as mediator of BK action on adenylate cyclase. In contrast, the stimulatory effect of BK on cAMP accumulation was completely abolished by two calmodulin antagonists, chlorpromazine and ophiobolin, suggesting an indirect, Ca2+/calmodulin-mediated effect of BK on the cAMP pathway. In addition, exposure of PC-12 cells to BK resulted in a translocation of the PKC isoforms alpha, delta, epsilon and zeta displaying different kinetics. The BK-induced translocations of the PCDs alpha and delta were rapid and biphasic, whereas the PKCs epsilon and zeta revealed a slower and slightly transient translocation in response to BK. The BK-elicited translocation of PKCepsilon, but not that of the PKCs alpha, delta and zeta, was prevented by two different inhibitors of adenylate cyclase, 2',5'-dideoxyadenosine and MDL-12,330A, as well as the PKA inhibitor adenosine 3':5'-monophosphothioate. These findings suggest that the BK-induced translocation of novel (n)PKCepsilon is mediated via the cAMP pathway. Since nPKCepsilon appears to regulate neurite outgrowth in PC-12 cells [Hundke, McMahon, Dadgar and Messing (1995) J. Biol. Chem. 270, 30134-30140] our results provide evidence for a novel signalling mechanism that might be involved in BK-induced neuronal differentiation of PC-12 cels.

Adenylate Cyclase Toxin↗
PubMed1987

Insulin inhibition of hepatic cAMP-dependent protein kinase: decreased affinity of protein kinase for cAMP and possible differential regulation of intrachain sites 1 and 2.

In hepatocytes stimulated with 8-bromo-cAMP, insulin decreases the affinity of the cAMP-dependent protein kinase for cAMP, shifting the Ka without affecting the Vmax activity. This occurs under conditions where cyclic adenine nucleotide concentrations are unchanged. We report here that glycogenolysis stimulated by 8-(4-chlorophenylthio)-cAMP, an analog with 100 times tighter affinity than cAMP for the protein kinase regulatory subunit, was only slightly antagonized by insulin. The tight binding of this analog appears to overcome the protein kinase affinity change induced by insulin. The relative importance of the two intrachain cAMP binding sites of the cAMP-dependent protein kinase regulatory subunit was investigated by using analogs with relative selectivity for each site. Analogs exhibiting preferential binding to site 2 were far less sensitive to insulin antagonism than were analogs binding preferentially at site 1 and less well at site 2. No other property of these analogs, including the rate of hydrolysis by phosphodiesterase, the IC50 for phosphodiesterase, the Ka for protein kinase, or the type I versus type II kinase specificity, could account for the ability of insulin to antagonize glycogenolysis stimulated by these analogs. These data indicate that insulin may act to decrease the affinity of protein kinases for cAMP through a possible regulation of intrachain site 2 binding.

Animals↗
PubMed1991

The promoter of the latency-associated transcripts of herpes simplex virus type 1 contains a functional cAMP-response element: role of the latency-associated transcripts and cAMP in reactivation of viral latency.

A 203-base-pair sequence 5' of the latency-associated transcripts (LATs) of herpes simplex virus type 1 contains a 7-base consensus sequence TGCGTCA that is identical to the cAMP-response element of the proenkephalin gene. This consensus sequence is at -38 relative to the putative 5' end of the LATs with a TATA box at the -24 position. In transient chloramphenicol acetyltransferase assays in rat pheochromocytoma (PC12) cells, this enhancer region stimulated gene expression up to 3-fold in the presence of dibutyryl cAMP, forskolin, nerve growth factor, or phorbol 12-myristate 13-acetate. Mutation of the cAMP-response element to TGCG-CAA resulted in a 4-fold reduction of basal activity and a complete loss of inducible stimulation. In DNA gel retardation assays, purified cAMP-response element-binding protein and a nuclear protein from PC12 cells were shown to bind specifically to this element. Furthermore, it was demonstrated that the reactivation of wild-type herpes simplex virus type 1 from dissociated latently infected murine trigeminal ganglia was significantly accelerated (P less than 0.005) by the addition of cAMP analogs or adenylate cyclase activators. However, these reagents did not accelerate reactivation of a deletion mutant that lacks the putative cAMP-response element-containing promoter region, transcriptional start site, and 1015 base pairs of the LATs. These studies demonstrate that the promoter region of the LATs contains a functional cAMP-response element and that expression of the LATs is likely controlled by second messenger signal transduction and imply a role for cAMP in triggering viral reactivation.

Animals↗
PubMed1998

Determination of the conformations of cAMP receptor protein and its T127L,S128A mutant with and without cAMP from small angle neutron scattering measurements.

Small angle neutron scattering (SANS) measurements were performed on solutions of cAMP receptor protein (CRP) and on solutions of the T127L,S128A double mutant of CRP (CRP*) in D2O K3PO4 buffer containing 0.5 M KCl, in the absence and presence of 3',5' cyclic adenosine monophosphate (cAMP). Energy-minimized structures of the CRP were calculated by minimization of the x-ray crystallographic structure of CRP in either the exclusively "closed" form where the alpha-helices of the carboxyl-terminal domain are folded close to the amino-terminal domain and in the exclusively "open" form where the alpha-helices of the carboxyl-terminal domain are folded away from the amino-terminal domain. Neutron scattering models show that the CRP SANS data follow closely the data curve predicted for unligated CRP in the open form, whereas the cAMP-ligated data are more in agreement with the data predicted for the minimized cAMP-ligated CRP structure in the closed form. Thus, it appears that CRP undergoes a conformational change from the open form to the closed form in solution upon ligation with cAMP. The SANS data from the CRP* and cAMP-ligated CRP* are coincidental, which implies that there is very little structural difference between the two species of CRP*. This is in agreement with in vivo results, which show that whereas CRP activates transcription in the cell only in the presence of cAMP, CRP* activates transcription in the absence of cAMP, implying that CRP* is already in the correct conformation for the activation of transcription.

Crystallography, X-Ray↗
PubMed1999

The type and the localization of cAMP-dependent protein kinase regulate transmission of cAMP signals to the nucleus in cortical and cerebellar granule cells.

cAMP signals are received and transmitted by multiple isoforms of cAMP-dependent protein kinases, typically determined by their specific regulatory subunits. In the brain the major regulatory isoform RIIbeta and the RII-anchor protein, AKAP150 (rat) or 75 (bovine), are differentially expressed. Cortical neurons express RIIbeta and AKAP75; conversely, granule cerebellar cells express predominantly RIalpha and RIIalpha. Cortical neurons accumulate PKA catalytic subunit and phosphorylated cAMP responsive element binding protein very efficiently into nuclei upon cAMP induction, whereas granule cerebellar cells fail to do so. Down-regulation of RIIbeta synthesis by antisense oligonucleotides inhibited cAMP-induced nuclear signaling in cortical neurons. Expression in cerebellar granule cells of RIIbeta and AKAP75 genes by microinjection of specific expression vectors, markedly stimulated cAMP-induced transcription of the lacZ gene driven by a cAMP-responsive element promoter. These data indicate that the composition of PKA in cortical and granule cells underlies the differential ability of these cells to transmit cAMP signals to the nucleus.

A Kinase Anchor Proteins↗
PubMed1999

Membrane-bound cAMP-dependent protein kinase controls cAMP-induced differentiation in PC12 cells.

The A126 cell line, a derivative of PC12, is defective in cAMP-induced transcription and does not differentiate in the presence of cAMP. In these cells overexpression of a cAMP-dependent protein kinase (PKA) anchor protein, AKAP75, and of the PKA catalytic subunit substantially increased the fraction of PKAII bound to the membrane, stimulated the transcription of cAMP-induced genes, and induced terminal differentiation. Conversely, wild type PC12 cells expressing a derivative of the AKAP75 protein, AKAP45, which binds the PKA regulatory subunits RII, but fails to locate them to the membranes, induced translocation of PKAII to the cytosol. These cells did not efficiently accumulate PKA catalytic subunit in the nuclei when stimulated with cAMP, did not transcribe cAMP-induced genes, and failed to differentiate when exposed to cAMP. These data indicate that membrane-bound PKA positively controls the transcription of cAMP-induced genes and differentiation in PC12 cells.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed2003

Characterization of a cAMP-stimulated cAMP phosphodiesterase in Dictyostelium discoideum.

A cyclic nucleotide phosphodiesterase, PdeE, that harbors two cyclic nucleotide binding motifs and a binuclear Zn(2+)-binding domain was characterized in Dictyostelium. In other eukaryotes, the Dictyostelium domain shows greatest homology to the 73-kDa subunit of the pre-mRNA cleavage and polyadenylation specificity factor. The Dictyostelium PdeE gene is expressed at its highest levels during aggregation, and its disruption causes the loss of a cAMP-phosphodiesterase activity. The pdeE null mutants show a normal cAMP-induced cGMP response and a 1.5-fold increase of cAMP-induced cAMP relay. Overexpression of a PdeE-yellow fluorescent protein (YFP) fusion construct causes inhibition of aggregation and loss of the cAMP relay response, but the cells can aggregate in synergy with wild-type cells. The PdeE-YFP fusion protein was partially purified by immunoprecipitation and biochemically characterized. PdeE and its Dictyostelium ortholog, PdeD, are both maximally active at pH 7.0. Both enzymes require bivalent cations for activity. The common cofactors Zn(2+) and Mg(2+) activated PdeE and PdeD maximally at 10 mm, whereas Mn(2+) activated the enzymes to 4-fold higher levels, with half-maximal activation between 10 and 100 microm. PdeE is an allosteric enzyme, which is approximately 4-fold activated by cAMP, with half-maximal activation occurring at about 10 microm and an apparent K(m) of approximately 1 mm. cGMP is degraded at a 6-fold lower rate than cAMP. Neither cGMP nor 8-Br-cAMP are efficient activators of PdeE activity.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed2005

Study of highly constitutively active mutants suggests how cAMP activates cAMP receptor protein.

The cAMP receptor protein (CRP) of Escherichia coli undergoes a conformational change in response to cAMP binding that allows it to bind specific DNA sequences. Using an in vivo screening method following the simultaneous randomization of the codons at positions 127 and 128 (two C-helix residues of the protein interacting with cAMP), we have isolated a series of novel constitutively active CRP variants. Sequence analysis showed that this group of variants commonly possesses leucine or methionine at position 127 with a beta-branched amino acid at position 128. One specific variant, T127L/S128I CRP, showed extremely high cAMP-independent DNA binding affinity comparable with that of cAMP-bound wild-type CRP. Further biochemical analysis of this variant and others revealed that Leu(127) and Ile(128) have different roles in stabilizing the active conformation of CRP in the absence of cAMP. Leu(127) contributes to an improved leucine zipper at the dimer interface, leading to an altered intersubunit interaction in the C-helix region. In contrast, Ile(128) stabilizes the proper position of the beta4/beta5 loop by functionally communicating with Leu(61). By analogy, the results suggest two direct local effects of cAMP binding in the course of activating wild-type CRP: (i) C-helix repositioning through direct interaction with Thr(127) and Ser(128) and (ii) the concomitant reorientation of the beta4/beta5 loop. Finally, we also report that elevated expression of T127L/S128I CRP markedly perturbed E. coli growth even in the absence of cAMP, which suggests why comparably active variants have not been described previously.

C-Reactive Protein↗
PubMed2006

Differential effects of Ca2+ and cAMP on transcription mediated by MEF2D and cAMP-response element-binding protein in hippocampal neurons.

In neurons, the second messengers Ca(2+) and cAMP are mediators of transcriptional responses that are important for the development and function of the nervous system. The pro-survival neuronal transcription factors cAMP-response elementbinding protein (CREB) and myocyte enhancer factor-2 (MEF2) both stimulate gene expression in response to activity-dependent increases in the concentration of intracellular Ca(2+) ions. CREB is also activated by increases in intracellular cAMP. Here we have investigated whether the MEF2 family member MEF2D, similar to CREB, is also activated by cAMP in hippocampal neurons. We have shown that, unlike CREB, MEF2D is not activated by agents that increase intracellular cAMP. Moreover, increases in cAMP inhibit Ca(2+)-activated MEF2D-mediated gene expression. We have also shown that cAMP inhibits Ca(2+)-induced nuclear export of the MEF2 co-repressor HDAC5 and prevents Ca(2+)-stimulated nuclear import of the MEF2 co-activator NFAT3/c4. Our results suggest that cAMP interferes with MEF2D-mediated gene expression at multiple levels by antagonizing the derepression of MEF2D by HDAC5 and by inhibiting recruitment of the co-activator NFAT.

Active Transport, Cell Nucleus↗
PubMed1985

The roles of cAMP and cAMP-dependent protein kinase in forskolin's actions on Y1 adrenocortical tumor cells.

The effects of forskolin on the regulation of steroidogenesis and growth were examined in the Y1 adrenocortical tumor cell line, and the roles of cAMP and cAMP-dependent protein kinase in these actions of forskolin were evaluated. Forskolin, like corticotropin, stimulated steroidogenesis 3-fold and inhibited growth by 90%. In mutants of the Y1 cell line harboring specific defects in cAMP-dependent protein kinase activity, the responses to forskolin were attenuated. The resistance of the protein kinase mutants to the diterpene was closely correlated with their resistance to corticotropin and with impaired responses of their protein kinases to cAMP. These results indicate that cAMP and cAMP-dependent protein kinase are obligatory components of forskolin's actions on Y1 adrenal cells. Forskolin, at concentrations which were approximately 100-times greater than those required to stimulate steroidogenesis, caused cAMP to accumulate. Apparently, only a small fraction of the cAMP generated in response to forskolin was required to stimulate steroidogenesis or inhibit growth.

Adenylyl Cyclases↗
PubMed1995

Differential effects on cAMP on the MAP kinase cascade: evidence for a cAMP-insensitive step that can bypass Raf-1.

Because cAMP exerts opposite effects on cell proliferation in different cell types, we undertook to study its effect on the mitogen-activated protein kinase (MAPK) pathway in three cell lines (Rat-1, Swiss-3T3, and COS-7) chosen for their different mitogenic responses to cAMP. We measured the effect of cAMP on MAPK, MEK, and Raf-1 activities after stimulation by agonists acting through a tyrosine kinase receptor (epidermal growth factor) or a G protein-coupled receptor (lysophosphatidic acid). In Rat-1 cells we found that cAMP strongly inhibited all three activities (MAPK, MEK, and Raf-1), in good agreement with its effect on cell proliferation in these cells. In Swiss-3T3 and COS-7 cells, on the contrary, cAMP did not inhibit epidermal growth factor- and lysophosphatidic acid-induced stimulation of MAPK and MEK activities, and even stimulated MAPK activity slightly on its own. Again these results are in good agreement with the proliferative effect of cAMP in Swiss-3T3 cells. Raf-1 activity on the hand, was inhibited by cAMP in Swiss-3T3 and COS-7 as it was in Rat-1 cells. This result indicates that signaling pathways in Swiss-3T3 and COS-7 cells can activate MEK and MAPK in a Raf-1-independent and cAMP-insensitive manner. Our results add to growing evidence for the existence of Ras- and/or Raf-1-independent pathways leading to MEK and MAPK activation.

3T3 Cells↗
PubMed2004

Exposure to cAMP and beta-adrenergic stimulation recruits Ca(V)3 T-type channels in rat chromaffin cells through Epac cAMP-receptor proteins.

T-type channels are expressed weakly or not at all in adult rat chromaffin cells (RCCs) and there is contrasting evidence as to whether they play a functional role in catecholamine secretion. Here we show that 3-5 days after application of pCPT-cAMP, most RCCs grown in serum-free medium expressed a high density of low-voltage-activated T-type channels without altering the expression and characteristics of high-voltage-activated channels. The density of cAMP-recruited T-type channels increased with time and displayed the typical biophysical and pharmacological properties of low-voltage-activated Ca(2+) channels: (1) steep voltage-dependent activation from -50 mV in 10 mm Ca(2+), (2) slow deactivation but fast and complete inactivation, (3) full inactivation following short conditioning prepulses to -30 mV, (4) effective block of Ca(2+) influx with 50 microM Ni(2+), (5) comparable permeability to Ca(2+) and Ba(2+), and (6) insensitivity to common Ca(2+) channel antagonists. The action of exogenous pCPT-cAMP (200 microM) was prevented by the protein synthesis inhibitor anisomycin and mimicked in most cells by exposure to forskolin and 1-methyl-3-isobutylxanthine (IBMX) or isoprenaline. The protein kinase A (PKA) inhibitor H89 (0.3 microM) and the competitive antagonist of cAMP binding to PKA, Rp-cAMPS, had weak or no effect on the action of pCPT-cAMP. In line with this, the selective Epac agonist 8CPT-2Me-cAMP nicely mimicked the action of pCPT-cAMP and isoprenaline, suggesting the existence of a dominant Epac-dependent recruitment of T-type channels in RCCs that may originate from the activation of beta-adrenoceptors. Stimulation of beta-adrenoceptors occurs autocrinally in RCCs and thus, the neosynthesis of low-voltage-activated channels may represent a new form of 'chromaffin cell plasticity', which contributes, by lowering the threshold of action potential firing, to increasing cell excitability and secretory activity during sustained sympathetic stimulation and/or increased catecholamine circulation.

Action Potentials↗
PubMed2004

Temporal characteristics of cAMP response element-mediated gene transcription: requirement for sustained cAMP production.

Many clinically used drugs are G-protein-coupled receptor (GPCR) antagonists and are given long-term to prevent receptor activation by endogenous agonists. Most GPCR antagonists are considered to have little agonist efficacy of their own. However, many beta antagonists do stimulate very small beta(2) adrenoceptor-mediated cAMP responses, but these responses become substantial at the level of cAMP response element (CRE)-gene transcription. Here, we compared the temporal characteristics of these beta(2) adrenoceptor-mediated cAMP and CRE-gene transcription responses with ligands of differing agonist efficacy. Within a minute, full agonists (e.g., isoprenaline) stimulated large increases in intracellular and exported cAMP. Very weak partial agonists (e.g., alprenolol) did not increase intracellular cAMP (only stimulating a small export). However, all agonists (regardless of efficacy) stimulated an increase in CRE-gene transcription after a 2-h incubation. An initial 30-min continual stimulation was required to initiate the process of CRE-gene transcription for all ligands. Longer agonist incubations resulted in larger gene transcription responses in a proportional manner for both weak and full agonists alike, and this was despite the lack of intracellular cAMP detection for the weaker ligands. Thus, the major initiator for CRE-gene transcription was not cAMP concentration or total quantity generated but a sustained turnover of intracellular cAMP and hence sustained stimulation of CREB phosphorylation. Thus, long-acting agonists and long-term treatments with very weak partial agonists (including many drugs classified previously as antagonists based on traditional second-messenger assays, e.g., several clinically used "beta-blockers") may cause more substantial gene transcription than previously believed.

Animals↗
PubMed1991

Cyclic adenosine 3',5'-monophosphate (cAMP)-dependent and cAMP-independent regulation of parathyroid hormone receptors on UMR 106-01 osteoblastic osteosarcoma cells.

The osteoblast-like cells, UMR 106-01, express PTH receptors that are coupled to adenylate cyclase. Recently, we reported the isolation of a UMR 106-01 subclone, UMR 4-7, that is stably transfected with a Zn(++)-inducible mutant of the regulatory subunit of protein kinase A. Incubation of UMR 4-7 cells with Zn++ renders the cells unresponsive to cAMP agonists. This subclone, therefore, seemed particularly suitable for studies of PTH receptor regulation. In UMR 106-01 cells, PTH receptors are strikingly down-regulated by pretreatment with 8-Br-cAMP or 3-isobutyl-1-methylxanthine for 2 days. In UMR 4-7 cells, this effect is totally prevented by prior and concurrent treatment with Zn++. Zn++ addition to UMR 106 cells does not modify these responses. Treatment with the PTH agonist [Nle8,18,Tyr34]bovine PTH(1-34)NH2 [(NlePTH(1-34)] also markedly down-regulates PTH receptors in UMR 106 cells, but this effect is only partially inhibited in Zn(++)-induced UMR 4-7 cells. At high doses, the PTH antagonist, [Nle8,18,Tyr34]bovine PTH(3-34)NH2 [NlePTH(3-34)] also (partially) reduces PTH receptor availability. Receptor regulation by NlePTH(3-34) is not blocked in the cAMP-resistant cells, however. Coincubation of submaximal doses of NlePTH(1-34) (1 nM) with NlePTH(3-34) (1 microM) reduces receptor availability more than when the cells are exposed to either ligand alone. This decrease is only partially inhibited in Zn(++)-induced UMR 4-7 cells. In contrast to its additive effect on receptor regulation, NlePTH(3-34) efficiently competes for binding to the PTH receptor in UMR 106-01 cells and antagonizes the stimulatory effects of NlePTH(1-34) on both intracellular cAMP accumulation and gene expression driven by a transiently transfected synthetic cAMP-responsive enhancer. In conclusion, homologous down-regulation of PTH receptors is mediated by activation of both cAMP-dependent (via protein kinase A) and cAMP-independent pathways. PTH activates both pathways, whereas the effect of NlePTH(3-34) appears to be exclusively cAMP-independent. These results give new insights into mechanisms of PTH receptor regulation.

1-Methyl-3-isobutylxanthine↗