Endogenous proteinases modulate the function of the β-adrenergic receptor-adenylate cyclase system

Photoaffinity labeling techniques have recently demonstrated that mammalian β₁- and β₂-adrenergic receptors reside on peptides of M_r 62 000–64 000. These receptor peptides are susceptible to endogenous metalloproteinases which produce peptides of M_r 30 000–55 000. Several proteinase inhibitors markedly attenuate this process, specifically EDTA and EGTA. In this study we investigated the functional significance of this proteolysis (and its inhibition) in the β₂-adrenergic receptor-adenylate cyclase system derived from rat lung membranes. Membrane preparations containing proteolytically derived fragments of the receptor of M_r 40000–55 000 are fully functional with respect to their ability to bind β-adrenergic antagonist radioligands such as [³H]dihydroalprenolol and β-adrenergic antagonist photoaffinity reagents such as p-azido-m-[^{125I]iodobenzylcarazolol}. They retain the ability to form a high-affinity, agonist-promoted, guanine nucleotide-sensitive complex thought to represent a ternary complex of agonist, receptor and guanine nucleotide regulatory protein. Nonetheless, after proteolysis, GTP is less able to revert this high-affinity receptor complex to one of lower affinity, and all aspects of adenylate cyclase stimulation are reduced. In addition, the functional integrity of the N protein in membranes prepared without proteinase inhibitors is reduced as assessed by reconstitution studies with the cyc[su− variant of S49 lymphoma cell membranes. These results suggest that endogenous proteolysis does not directly impair the ability of β-adrenergic receptors to either bind ligands or interact with the guanine nucleotide regulatory protein. However, they imply that endogenous proteolysis likely impairs the functionality of other components of the adenylate cyclase system, such as the nucleotide regulatory protein.     

Insulin desensitized beta 1-adrenergic receptor-mediated stimulation of adenylyl cyclase in SK-N-MC cells.

Receptor cross-talk is an emerging field which investigates cross-regulation between distinct classes of receptors. In the present work, we investigated the influence of activating the insulin receptor, a tyrosine kinase receptor, on beta-agonist activation of adenylyl cyclase, which is mediated by a G protein-linked receptor. Treatment of SK-N-MC neuroepithelioma cells with insulin generated a marked attenuation of beta 1-adrenergic receptor-mediated stimulation of adenylyl cyclase. This effect required nanomolar concentrations of insulin, occurred within minutes of exposure of these cells to insulin, and did not result from down-regulation of beta-adrenergic receptors. Insulin alone reduced the maximal isoproterenol-mediated stimulation of adenylyl cyclase by 50%, while the co-addition of the phosphatase inhibitor sodium vanadate increased the magnitude of insulin inhibition to 90%. Insulin provides an additional avenue for heterologous desensitization of beta-adrenergic receptors and their transmembranal signalling pathway.     

Gβγ that interacts with adenylyl cyclase in opioid tolerance originates from a Gs protein

We previously demonstrated that chronic morphine induces a change in G protein coupling by the mu opioid receptor (MOR) from Gi/o to Gs, concurrent with the instatement of an interaction between Gβγ and adenylyl cyclase types II and IV. These two signaling changes confer excitatory effects on the cell in place of the typical inhibition by opioids and are associated with morphine tolerance and dependence. Both signaling changes and these behavioral manifestations of chronic morphine are attenuated by cotreatment with ultra‐low‐dose naloxone. In the present work, using striatum from chronic morphine‐treated rats, we isotyped the Gβ within Gs and Go heterotrimers that coupled to MOR and compared these to the Gβ isotype of the Gβγ that interacted with adenylyl cyclase II or IV after chronic morphine treatment. Isotyping results show that chronic morphine causes a Gs heterotrimer associated with MOR to release its Gβγ to interact with adenylyl cyclase. These data suggest that the switch to Gs coupling by MOR in response to chronic morphine, which is attenuated by ultra‐low‐dose opioid antagonist cotreatment, leads to a two‐pronged stimulation of adenylyl cyclase utilizing both Gα and Gβγ subunits of the Gs protein novel to this receptor. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006     

β3-Adrenergic activation of adenylyl cyclase in mouse white adipocytes: modulation by GTP and effect of obesity

Lipolysis and adenylyl cyclase (AC) activation in response to β-adrenergic agents are abnormally low in white epididymal adipose tissue (WAT) of the ob/ob mouse. The abundance of G-proteins (G_sα and G_iα) linked to AC is also abnormally low. By contrast, β-adrenergic receptor (β-AR) levels were previously found to be normal in WAT and elevated in liver. The relative importance of various forms of the β-AR in mouse WAT was reassessed in view of the discovery of the β₃-AR. The results show that (1) the β₃-AR is mainly responsible for AC activation in lean-mouse WAT; (2) the β₃-AR is only partly responsible for AC activation in obese mouse WAT; and (3) GTP modulates β₃—-but not β₁—-or β₂-AR activation of AC in a biphasic manner. Therefore, the β₃-AR appears responsible for the well-known bimodal effect of GTP on β-adrenergic receptor-mediated AC activity in WAT.     

Type-specific regulation of adenylyl cyclase. Selective pharmacological stimulation and inhibition of adenylyl cyclase isoforms

Crystallographic studies have elucidated the binding mechanism of forskolin and P-site inhibitors to adenylyl cyclase. Accordingly, computer-assisted drug design has enabled us to identify isoform-selective regulators of adenylyl cyclase. After examining more than 200 newly synthesized derivatives of forskolin, we found that the modification at the positions of C6 and C7, in general, enhances isoform selectivity. The 6-(3-dimethylaminopropionyl) modification led to an enhanced selectivity for type V, whereas 6-[N-(2-isothiocyanatoethyl) aminocarbonyl] and 6-(4-acrylbutyryl) modification led to an enhanced selectivity for type II. In contrast, 2'-deoxyadenosine 3'-monophosphate, a classical and 3'-phosphate-substituted P-site inhibitor, demonstrated a 27-fold selectivity for inhibiting type V relative to type II, whereas 9-(tetrahydro-2-furyl) adenine, a ribose-substituted P-site ligand, showed a markedly increased, 130-fold selectivity for inhibiting type V. Consequently, on the basis of the pharmacophore analysis of 9-(tetrahydro-2-furyl) adenine and adenylyl cyclase, a novel non-nucleoside inhibitor, 2-amino-7-(2-furanyl)-7,8-dihydro-5(6H)-quinazolinone (NKY80), was identified after virtual screening of more than 850,000 compounds. NKY80 demonstrated a 210-fold selectivity for inhibiting type V relative to type II. More importantly, the combination of a type III-selective forskolin derivative and 9-(tetrahydro-2-furyl) adenine or NKY80 demonstrated a further enhanced selectivity for type III stimulation over other isoforms. Our data suggest the feasibility of adenylyl cyclase isoform-targeted regulation of cyclic AMP signaling by pharmacological reagents, either alone or in combination.     

Appearance of β-adrenergic response of adenylate cyclase during the induction of differentiation in cell cultures

Chemical inducers of differentiation, such as dimethylsulfoxide, N-methylacetamide and sodium butyrate, induce various kinds of differentiated characteristics in tumor cell lines as shown by numerous studies. In this study we showed that one common property of this group of chemicals is their ability to induce β-adrenergic response of adenylate cyclase in cell cultures, including Friend erythroleukemic cells, HeLa cells and rat fetal hepatocytes. This induction appears to be an early event in the differentiation, since it precedes the appearance of other differentiated characteristics, such as hemoglobin synthesis in erythroleukemic cells. This induced hormone response may contribute to the regulation of subsequent steps of differentiation.     

Visualization of the turkey erythrocyte β-adrenergic receptor

We have recently described the affinity chromatography purification of the turkey erythrocyte β-adrenergic receptor. The minute amounts obtained initially precluded extensive biochemical characterization. To improve the yield of the receptor, the erythrocyte membranes have been prepared by a new method. This procedure resulted in a 10-fold higher receptor density in comparison with the membrane preparation used previously. The new membranes also contained a catecholamine-sensitive guanine triphosphatase and an adenylate cyclase sensitive to Gpp(NH)p and l-epinephrine. Solubilization by a double digitonin extraction resulted in a preparation containing 4–6 pmoles of ³H-dihydroalprenolol binding sites per mg of membrane protein. A single step of affinity chromatography on alprenolol-sepharose of the soluble digitonin extract resulted in an additional 1,000-fold purification of the receptor. The overall purification factor was 20,000 relative to the binding activity of the crude membrane preparations. Electrophoresis in SDS-polacrylamide of iodinated purified β-receptors revealed, after autoradiography, the presence of four major components. Three of these, corresponding to molecular weights of 170,000, 33,000, and 30,000, respectively, were not affected by reduction with β-mercaptoethanol and were not observed when the digitonin extracts were loaded on the affinity gel in the presence of an excess of l-propranolol. A fourth 52,000-dalton component (60,000 daltons after reduction with β-mercaptoethanol) remained apparent even when affinity purification was prevented by addition of l-propranolol. Our results suggest that the β-adrenergic receptor is composed of at least three subunits that interact by noncovalent bonds.     

Structural classification of αββ and ββα supersecondary structure units in proteins

We present a fully automatic structural classification of supersecondary structure units, consisting of two hydrogen-bonded β strands, preceded or followed by an α helix. The classification is performed on the spatial arrangement of the secondary structure elements, irrespective of the length and conformation of the intervening loops. The similarity of the arrangements is estimated by a structure alignment procedure that uses as similarity measure the root mean square deviation of superimposed backbone atoms. Applied to a set of 141 well-resolved nonhomologous protein structures, the classification yields 11 families of recurrent arrangements. In addition, fragments that are structurally intermediate between the families are found; they reveal the continuity of the classification. The analysis of the families shows that the α helix and β hairpin axes can adopt virtually all relative orientations, with, however, some preferable orientations; moreover, according to the orientation, preferences in the left/right handedness of the α–β connection are observed. These preferences can be explained by favorable side by side packing of the α helix and the β hairpin, local interactions in the region of the α–β connection or stabilizing environments in the parent protein. Furthermore, fold recognition procedures and structure prediction algorithms coupled to database-derived potentials suggest that the preferable nature of these arrangements does not imply their intrinsic stability. They usually accommodate a large number of sequences, of which only a subset is predicted to stabilize the motif. The motifs predicted as stable could correspond to nuclei formed at the very beginning of the folding process. Proteins 30:193–212, 1998. © 1998 Wiley-Liss, Inc.     

An avidin-biotinyl-propranolol complex for β-adrenergic receptor characterization

The synthesis of biotinyl-hexaglycyl-NEDA (abbreviation:BGN), a biotinyl derivative of propranolol, is described. This bifunctional molecule binds with high affinity to the biotin-binding protein, avidin. The duck erythrocyte was used as a model β-receptor system. Formation of an avidin-BGN-β-receptor complex was demonstrated in intact erythrocytes, in erythrocyte ghosts, and in the digitonin-solubilized β-receptor. The avidin-BGN complex will be used for localization and purification of the β-receptor.     

Inositol hexakisphosphate increases L-type Ca2+ channel activity by stimulation of adenylyl cyclase.

Inositol hexakisphosphate (InsP6) is a most abundant inositol polyphosphate that changes simultaneously with inositol 1,4,5-trisphosphate in depolarized neurons. However, the role of InsP6 in neuronal signaling is unknown. Mass assay reveals that the basal levels of InsP6 in several brain regions tested are similar. InsP6 mass is significantly elevated in activated brain neurons and lowered by inhibition of neuronal activity. Furthermore, the hippocampus is most sensitive to electrical challenge with regard to percentage accumulation of InsP6. In hippocampal neurons, InsP6 stimulates adenylyl cyclase (AC) without influencing cAMP phosphodiesterases, resulting in activation of protein kinase A (PKA) and thereby selective enhancement of voltage-gated L-type Ca2+ channel activity. This enhancement was abolished by preincubation with PKA and AC inhibitors. These data suggest that InsP6 increases L-type Ca2+ channel activity by facilitating phosphorylation of PKA phosphorylation sites. Thus, in hippocampal neurons, InsP6 serves as an important signal in modulation of voltage-gated L-type Ca2+ channel activity.     

Regulation of adenylyl cyclase activity by beta-adrenergic agonists in a desensitization-resistant mutant cell line.

Mutant clones resistant to ACTH-induced desensitization of adenylyl cyclase (Y1DR) were previously isolated from the Y1 mouse adrenocortical tumor cell line. In this study, both parental Y1 cells (Y1DS) and a Y1DR mutant were transfected with a gene encoding the mouse beta 2-adrenergic receptor, and transfectants isolated from both Y1DS and Y1DR cells were shown to express beta 2-adrenergic receptors. These transfectants responded to the beta-adrenergic agonist isoproterenol with increases in adenylyl cyclase activity and steroidogenesis and changes in cell shape. The transfectants were analyzed to determine whether the Y1DR mutation was specific for ACTH-induced desensitization of adenylyl cyclase or also affected desensitization of adenylyl cyclase via the beta 2-adrenergic receptor. Treatment of intact Y1DS transfectants with isoproterenol caused a rapid desensitization of the adenylyl cyclase system to further stimulation by the beta-adrenergic agonist. Treatment of intact cells with isoproterenol did not affect ACTH-stimulated adenylyl cyclase activity, indicating that desensitization was agonist specific or homologous. Y1DR transfectants were resistant to the desensitizing effects of isoproterenol in intact cells as well as in cell homogenates. These results indicate that the mutation in Y1DR transfectants affects a component that is common to the pathways of isoproterenol-induced desensitization and ACTH-induced desensitization of adenylyl cyclase. As determined using the hydrophilic beta-receptor antagonist CGP-12177, isoproterenol caused a rapid sequestration of cell surface receptors in both Y1DS and Y1DR transfectants. From these results we infer that the DR phenotype does not arise from mutations affecting receptor sequestration and that receptor number does not limit the response to isoproterenol in these transfectants.     

Evidence for dual coupling of the murine luteinizing hormone receptor to adenylyl cyclase and phosphoinositide breakdown and Ca2+ mobilization. Studies with the cloned murine luteinizing hormone receptor expressed in L cells.

The murine receptor for luteinizing hormone (LHR) was cloned and expressed in L cells. This LHR (mature protein of 674 amino acids) is very similar to that of the rat (same length, 36 amino acid differences) but differs significantly more from that of man (673 amino acids, 109 differences). Expression of the murine LHR in L cells led to the appearance of binding sites for human chorionic gonadotropin (hCG) with a Kd of 150 pM and an LH- and hCG-stimulable adenylyl cyclase activity (EC50 = 50-100 pM hCG). Upon labeling pools of phosphoinositides with [3H]myo-inositol, L cells expressing the murine LHR responded to hCG with an increase in their rate of phosphoinositide hydrolysis (EC50 = 2,400 pM hCG). This was accompanied by an increase in intracellular Ca2+ [( Ca2+]i), as determined by the Fura2 method. This increase in [Ca2+]i in response to hCG was dependent on the LHR, for HCG did not affect [Ca2+]i in L cells not expressing the LHR. The effect was not due to the cAMP-forming activity of the LH receptor, for neither forskolin nor prostaglandin E1, which both increase cAMP levels in L cells, had a similar effect in either control or LHR-expressing cells and isoproterenol had no effect in L cells expressing a functionally active hamster beta-adrenergic receptor. The effect was also not due to overexpression of a Gs-coupled receptor, for L cells expressing 8-fold higher levels of the human V2 vasopressin receptor did not mimic the Ca(2+)-mobilizing response of the LH receptor. We conclude that the LH receptor has the capability of activating two intracellular signaling pathways: one leading to stimulation of adenylyl cyclase and resulting in increases in cAMP and a second leading to stimulation of phospholipase C and resulting in formation of inositol phosphates and elevations in [Ca2+]i. These data correlate positively with and provide a mechanistic explanation for previous reports on the ability of hCG to mobilize phosphoinositides and increasing [Ca2+]i in luteal and granulosa cells (e.g. Davis, J. S., West, L. A., and Farese, R. V. (1984) J. Biol. Chem. 259, 15028-15034).     

Effects of luteinizing hormone-releasing hormone agonist and calcium upon adenylyl cyclase activity of human corpus luteum membranes

We have investigated the ability of the agonist analog of luteinizing hormone-releasing hormone (LH-RH), D-Trp6-LH-RH (LH-RHa), and of CaCl2 to inhibit directly gonadotropin stimulation of adenylyl cyclase in a cell-free system prepared from human corpus luteum. In the presence of a submaximally effective concentration of hCG, addition of 10(-5)M final concentration of LH-RHa did not alter the gonadotropin-stimulated enzyme activity, nor did LH-RHa alone show any effect upon basal levels of the enzyme. The failure to inhibit adenylyl cyclase would indicate that the LH-RHa does not affect gonadotropin receptor binding or cAMP synthesis and/or degradation in this membrane system, suggesting that the luteolytic effects of LH-RH are unlikely to involve a direct antigonadotropic activity at the level of the human corpus luteum. In great contrast to LH-RHa, addition of CaCl2 resulted in a dose-dependent inhibition of hCG-stimulable adenylyl cyclase. Thus, in the presence of either a maximally or submaximally effective concentration of hCG, inhibition was significant at 0.5 mM CaCl2 added in excess of ATP (2 mM) and EDTA (1 mM), being about 90% upon addition of 2.5 mM CaCl2. We also found that calcium reduced enzyme stimulation by forskolin and the GTP analog, guanyl 5'-yl imidodiphosphate [GMP-P(NH)P] in a dose-related manner and that activation by NaF was less sensitive to inhibition by calcium. Accordingly, at 2.5 mM CaCl2, guanyl nucleotide and forskolin stimulations were inhibited 96% and 86%, respectively, while NaF stimulation was reduced by 40%. Because previous studies have shown that calcium does not impair gonadotropin binding activity, the calcium-dependent inhibition of gonadotropin responsiveness reported here would imply an alteration in the functional coupling of the components of the luteal adenylyl cyclase system. These data suggest that calcium may play a role in the regulation of gonadotropin action in the human corpus luteum.     

Lithium induces follicular atresia in rat ovary through a GSK‐3β/β‐catenin dependent mechanism

Lithium chloride (LiCl) is a drug used to treat bipolar disorder, but has side effects in the female reproductive system. Although lithium is known to decrease folliculogenesis and induce follicular atresia in rodent ovaries, its cellular and molecular effects in the ovary have not yet been addressed. To investigate these effects, 23‐day‐old immature female rats were injected with 10 IU pregnant mare serum gonadotropin (PMSG), followed by injections of 250 mg/kg LiCl every 12 hr for four doses. Ovaries were removed 40 and 48 hr after PMSG administration and prepared for histology, immunohistochemistry, Western blotting, and DNA laddering analysis. Our results showed that in the ovaries of LiCl‐treated rats, few antral but more atretic follicles were present compared to those of the control rats. The induction of atresia by LiCl was further confirmed by the presence of DNA fragmentation, accompanied by a reduced level of 17β‐estradiol in the serum. At the cellular level, lithium significantly decreased the number of proliferating cell nuclear antigen (PCNA)‐positive cells and conversely increased the number of TUNEL‐positive cells in the granulosa layer of the antral follicles. At the molecular level, lithium increased the level of phosphorylated glycogen synthase kinase‐3β, and unexpectedly decreased the expression of active (stabilized) β‐catenin. Altogether, our results indicate that lithium disrupts the balance between proliferation and apoptosis in granulosa cells, leading to follicular atresia possibly through the reduction in both the stabilized β‐catenin and 17β‐estradiol synthesis. Mol. Reprod. Dev. 80: 286–296, 2013. © 2013 Wiley Periodicals, Inc.     

Role of 5'-guanylylimidodiphosphate in the activation of adenylyl cyclase by parathyroid hormone.

We have studied the effects of guanylylimidodiphosphate (Gpp(NH)p), an analogue of GTP, on the stimulation of renal cortical adenylyl cyclase by bovine parathyroid hormone (bPTH, or bPTH-(1-84)). Incubation of canine renal membranes with bPTH-(3-34), a specific antagonist of parathyroid hormone, in either the presence or absence of Gpp(NH)p, prevented subsequently added bPTH-(1-84) from stimulating adenylyl cyclase. The addition of the antagonist to a cyclase system previously activated by both bPTH-(1-84) and Gpp(NH)p, however, produced no inhibition of enzyme activity. Removal of bPTH by washing the membranes virtually abolished activity, but washing after addition of bPTH plus Gpp(NH)p did not prevent continued accumulation of cAMP. The persistence of the activity of the enzyme brought about by the addition of Gpp(NH)p plus bPTH, despite washing or addition of specific inhibitor of bPTH action, indicates that the activity of the hormone-specific adenylyl cyclase in membrane suspensions is independent of cintinuous occupancy of the peptide-hormone receptor by bPTH in the presence of the guanyl-nucleotide analogue.