Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1384
The Role of Protein Kinase C in the Extracellular Ca2+-regulated Secretion of Parathyroid Hormone
BY AMOS M. SAKWE
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:00 !8!69#9" :0 ;6$$#6"9 6; 6#" 9 )& DD ,1,D G H 6#" 9+ In memory of my father, Henry Sakwe Itoe This dissertation is based on the following manuscripts, referred to in the text by their roman numerals.
I Sakwe AM, Engström Å, Larsson M, Rask L. (2002) Biosynthesis and secretion of parathyroid hormone are sensitive to proteasome inhibitors in dispersed bovine parathyroid cells. J Biol Chem. 277,17687-17695.
II Sakwe AM, Larsson M, Rask L. (2004) Involvement of protein kinase C- alpha and -epsilon in extracellular Ca2+ signalling mediated by the calcium sensing receptor. Exp. Cell Res. 297, 560-573.
III Sakwe AM, Rask L, Gylfe E. (2004) Protein kinase C modulates agonist-sensitive release of Ca2+ from internal stores in HEK293 cells over-expressing the calcium sensing receptor. (Manuscript)
IV Sakwe AM, Akusjärvi G, Rask L. (2004) Physiological activation of PKC-alpha via the calcium sensing receptor is not required for the Ca2+-regulated secretion of parathyroid hormone in dispersed bovine parathyroid cells. (Manuscript)
Reprints were made with permission from the publishers. Contents
Introduction...... 7 Biosynthesis and secretion of parathyroid hormone in the parathyroid glands...... 8 Parathyroid glands and parathyroid hormone...... 8 Biosynthesis and maturation of PTH...... 8 Detection and Measurement of secreted PTH...... 10 Regulation of extracellular Ca2+ concentration ...... 10 Extracellular Ca2+ sensing and signalling ...... 12 The calcium sensing receptor...... 12 Protein kinase C ...... 15 Regulation of Parathyroid Hormone Secretion...... 19 Stimulus-secretion coupling...... 19 The molecular events during exocytosis ...... 19 Regulation of PTH secretion by intracellular degradation...... 21 Mechanisms of action of pharmacologically active agents on PTH secretion ...... 22 Results and Discussion ...... 23 Objectives and experimental approach...... 23 Summary of our observations...... 24 Rationale for Papers II, II & IV...... 25 Discussion ...... 28 Acknowledgements...... 32 References...... 33 Abbreviations
ADH Autosomal dominant hypercalcemia CaR extracellular Ca2+ sensing receptor CTD cytosolic domain DAG Diacylglycerol DN PKC Dominant negative mutant protein kinase C ECD Extracellular domain ERK Extracellular signal-regulated protein kinase FBS Fetal bovine serum FHH Familial hypocalciuric hypercalcemia GPCR G protein coupled receptor IP3 Inositol-1,4,5-triphosphate MEK MAP kinase kinase NSHPT neonatal severe hyperparathyroidism PKC Protein kinase C PKM Protein kinase M (catalytic fragment of PKC) PT Parathyroid PTH Parathyroid hormone RT-PCR Reverse transcriptase polymerase chain reaction SNAP Soluble N-ethylmaleimide attachment protein SNARE Soluble N-ethylmaleimide attachment factor receptor TMD Transmembrane domain TPA 12-O-tetradecanoylphorbol-13-acetate wt PKC wild type protein kinase C Introduction
Parathyroid (PT) cells are probably the only cell type in mammals that can synthesize and secrete parathyroid hormone (PTH), a bioactive peptide hormone that is directly involved in the regulation of the concentration of 2+ 2+ extracellular Ca (>Ca @o). In these cells, secretion of the hormone is 2+ inhibited at high >Ca @o. A number of theories were postulated to explain 2+ this inverse relationship between PTH secretion and >Ca @o. These include regulated translation of PTH mRNA [1-3], regulated intracellular degradation of the mature hormone in the secretory pathway [4, 5] and/or direct regulation of the secretory process as such by changes in cytosolic 2+ 2+ 2+ Ca (>Ca @i). The cloning and characterisation of the extracellular Ca sensing receptor (CaR) in 1993 [6] provided more insight into the possible 2+ mechanisms in that activation of the receptor at high >Ca @o led to an 2+ 2+ increase of the intracellular Ca concentration (>Ca @i) and theoretically, activation of protein kinase C (PKC). However, studies on the stimulus- secretion coupling based on changes in intracellular Ca2+ concentration produced contradictory observations [7]. Similarly, it was hard to reconcile 2+ any role of PKC in this process since high >Ca @o-induced activation of the CaR and consequently, PKC suppressed PTH secretion, but treatment of 2+ these cells with TPA, a potent PKC activator, uncoupled the high >Ca @o- induced suppression of PTH secretion [8-12]. The studies reported herein addressed these discrepancies in order to contribute to the elucidation of the 2+ molecular mechanism(s) by which slight changes in the >Ca @o modulate the secretion of PTH in PT cells with emphasis on the role of PKC as a target downstream of the CaR.
7 Biosynthesis and secretion of parathyroid hormone in the parathyroid glands
Parathyroid glands and parathyroid hormone Parathyroid (PT) glands which lie adjacent to or embedded in the thyroid glands were first described by Ivar Sandström in 1879 in humans [13]. These glands are small yellow-brown, oval bodies weighing between 50 and 300 mg, and ranging between 3 and 8 mm in thickness. In humans, there are usually four PT glands: the two upper ones are almost always located near or at the posterior surfaces of the lower poles of the thyroid gland. The lower two glands may lie either embedded into the thyroid, outside the thyroid or adjacent to the thymus [13-15]. In the studies described herein, the upper two PT glands were easily identified and isolated from cows of different ages and both sexes at the local slaughterhouse. The parenchyma of normal human PT glands comprises chief cells, large clear cells, transitional-oxyphil cells, and oxyphil cells [16]. The morphology of PT glands varies with 2+ sustained changes in >Ca @o [17, 18]. With age, the glands contain a variable amount of interstitial fat and the ratio of PT chief cells to oxyphilic cells decreases [19]. The most abundant and important cell type in these glands is the PT chief cell which is probably the only cell type in higher animals equipped with the machinery to synthesize and secrete PTH. The less abundant oxyphilic cells arise from PT chief cells and play little or no role in the biosynthesis and secretion of PTH.
Biosynthesis and maturation of PTH The gene coding for human PTH is located on chromosome 11 [20-22] and comprises 3760 bp [23, 24] while the bovine gene consists of 3154 bp [25]. In both species, the gene contains three exons. The complete coding sequence (mRNA) of the human hormone is 828 bp while that for the bovine hormone is 699 bp long [24, 26]. Like many secreted proteins, PTH is synthesized as preproPTH containing a signal peptide of 25 amino acid residues. The primary precursor also contains a pro-segment of six essentially basic amino acid residues and the remaining carboxy terminal 84
8 amino acid residues constitute the mature bioactive PTH. The primary precursor undergoes two successive proteolytic cleavages as it progresses along the secretory pathway [27, 28]. The first cleavage catalysed by a signal peptidase, occurs either co-translationally or soon after translocation into the endoplasmic reticulum [29, 30] to produce proPTH, which is the predominating form in the early portion of the secretory pathway. The second cleavage that occurs in the trans-Golgi network is thought to be effected by a furin-like pro-hormone convertase [31, 32] to yield mature bioactive PTH, which is thereafter either packaged into storage granules or secretory granules. The latter progress to the plasma membrane for exocytosis and export of the hormone (Fig. 1).
Fig. 1. Biosynthesis and transit of PTH in the secretory pathway (adapted from Kemper et al., 1975)
Based on data using radiolabelling and immunoprecipitation techniques, the formation of proPTH is thought to occur within 1 min, while the secretion of de novo synthesized PTH occurs within 30 min (Fig. 1) following the formation of preproPTH. The bulk of secreted PTH is believed to originate from the newly synthesized pool [33-35]. The N-terminal portion of PTH (PTH1-34) is believed to possess most of the biological activity while the role of the C-terminal portion, which is also found in circulation, is still not clear. A cross-section of a parathyroid chief cell reveals that under normal growth conditions, large amounts of the mature hormone are stored in membrane-bound storage granules [36, 37].
9 Detection and Measurement of secreted PTH PTH secreted from PT chief cells is released into the extracellular space from which it enters the blood stream to be transported to its target organs. Several immuno-radiometric assays (IRMA) and enzyme-linked immunosorbent assays (ELISA) have been used to quantitatively estimate secreted PTH. These are based on specific antibodies to detect either the full- length hormone [38, 39] or its fragments [40, 41]. Since C-terminal fragments can be detected along side the full-length hormone, there is a greater chance of erroneous estimation of the secreted intact hormone in cell/organ culture or body fluids using assays based on the C-terminal fragment. Assays developed by Immutopics Inc. for the estimation of secreted PTH are sandwich IRMA or ELISA that detect intact PTH based on the detection of the N-terminal fragment of the whole molecule using two affinity purified antibodies to this fragment [42, 43]. Since the N-terminal fragment is rapidly degraded upon proteolysis of the mature hormone in vivo and is not secreted, it will not interfere with the quantitation of the intact PTH in PT cell culture media or body fluids by this assay. Our PTH measurements were carried out using this assay since we were interested in the secretion of intact PTH rather than its proteolytic products.
Regulation of extracellular Ca2+ concentration In the body, Ca2+ is distributed into three major pools: Inside cells, Ca2+ is found stored in the ER and mitochondria (internal stores) as well as in the cytosol as free intracellular Ca2+. The latter, which normally fluctuates around 100 nM in resting cells, may increase up to 1 PM as a result of release from internal stores or influx of Ca2+ from the extracellular compartment. In the body fluids or extracellular milieu, Ca2+ is found either bound to proteins (50%), diffusible but unionised (5%) or as free ionised Ca2+ (45%). In bone, Ca2+ exists predominantly (99%) as crystalline salts of Ca2+ and phosphate denoted hydroxyapatites deposited on the ground substance of the bone but also, as free Ca2+ (1 %) in equilibrium with that in the surrounding EC compartment. Given the importance of Ca2+ in almost every cellular process, it is not surprising that the homeostatic control of Ca2+ is one of the most elaborate regulatory mechanisms in the body [44]. This is assured by the effects of the calciotropic hormones viz, vitamin D, calcitonin and PTH. PTH is the primary regulator of EC fluid or plasma ionised calcium (Ca2+) in higher animals. Together with calcitonin and 1, 25 dihydroxyvitamin D3 (1, 25(OH)2D3) PTH tightly regulates the concentration of extracellular fluid 2+ 2+ Ca within 1.1-1.3 mM [44-47]. When the >Ca @o decreases, secretion of PTH is stimulated and this has the following effects: In the kidneys, it
10 stimulates the rate of reabsorption of not only calcium but also Mg2+ and + + + H3O . PTH also inhibits renal reabsorption of phosphate, Na , K and amino acids in the distal renal tubules, substances which in most part are excreted in urine. In bone, secreted PTH enhances the activity of mainly osteoclasts leading to the mobilization of Ca2+ from bone. Sustained secretion of PTH also stimulates the 1-D-hydroxylase activity in the proximal tubules, which leads to increased synthesis of the active vitamin D metabolite 1, 25(OH)2D3 from 25(OH)D3. Active 1, 25(OH)2D3 stimulates the intestinal absorption of dietary calcium and has long-term effects on bone calcium turnover. Therefore, in hypocalcemia, the actions of PTH on these organs directly or 2+ indirectly lead to regeneration of >Ca @o to near normal levels. Calcitonin is a calciotropic hormone synthesized and secreted by thyroid C cells or parafollicular cells. Its secretion is inhibited by hypocalcemia but stimulated by hypercalcemia. The major role of calcitonin is to inhibit osteoclastic bone resorption and to retard the transport of Ca2+ from bone to the extracellular fluid and blood and by these actions, which are opposite to 2+ those of PTH, the elevated >Ca @o decreases to near physiological concentrations.
11 Extracellular Ca2+ sensing and signalling
The calcium sensing receptor Structure and tissue distribution The primordial role of the PT cell in Ca2+ homeostasis is to secrete PTH and 2+ this in turn is tightly regulated by >Ca @o. This regulated secretion of PTH therefore implied the existence of an efficient mechanism(s) for the sensing 2+ of changes in >Ca @o to generate the ultimate PTH secretory response. The existence of such a regulatory mechanism became apparent with the findings that PT cell membranes could bind Ca2+ [48] and that in PT cells, high Ca2+ and other divalent ions caused the accumulation of inositol triphosphate [49, 50] and DAG [51]. An even more important observation with respect to this 2+ 2+ was that high >Ca @o caused a similar change in the >Ca @i [52, 53]. The cloning and characterisation of the parathyroid extracellular Ca2+ sensing receptor (CaR) in 1993 [6] confirmed these hypotheses. Subsequent identification of the receptor in the kidney [54] and in several other human tissues [55-62] as well as in the tissues of organisms as distant from humans as birds, fishes and amphibians [63-68] demonstrated that such a Ca2+ sensing and/or homeostatic apparatus is ubiquitous. Its role in tissues not involved in Ca2+ and mineral homeostasis such as the brain [63] is not yet clear and suggests that there may be several CaRs [69]. The CaR gene is located on human chromosome 3q13.3-21, rat chromosome 11 or mouse chromosome 16 [70]. It encodes a seven transmembrane heterotrimeric G protein-coupled cell-surface receptor (GPCR) of about 1085 (bovine PT) or 1078 (human kidney) amino acid residues [6, 54]. The CaR belongs to family C (III) of GPCRs and comprises a large extracellular domain (ECD) of about 600 amino acid residues that is separated from the small cytosolic domain (CTD) of about 200 amino acid residues by the seven transmembrane-spanning domain (TMD). The ligand binding sites are located in the ECD and to the EC portions of the TMD [71]. The ECD is also the region where the receptor dimerizes [72] and it is glycosylated to varying extents [73-75]. The CTD and the intracellular portions of the TMD are the regions that interact with cytosolic components including heterotrimeric G proteins and the scaffolding protein filamin [76, 77]. The CTD contains at least five putative PKC phosphorylation sites including Thr888 [78, 79].
12 Molecular signalling from the CaR The CaR responds to a large number of ligands including divalent cations such as Ca2+, Mn2+, Co2+, Ni2+ Ba2+ etc.; trivalent ions like Gd3+, La3+; polyamines like spermine and spermidine; antibiotics like neomycin and L- amino acids [6, 80-83]. Devoid of kinase activity, it relies on its interaction with heterotrimeric G proteins to influence different cellular processes in several tissues. These include Ca2+ and mineral homeostasis, secretion, cell growth and proliferation, differentiation and apoptosis [84-86]. Like the GPCRs of this family, binding of agonist to this receptor activates different classes of heterotrimeric G proteins [87]. These include heterotrimeric proteins of the class GDq and GD11, which activate phospholipase C-E [88, 89], GD12/13, which leads to the activation of phospholipase D [90] and GDi/o, which inhibits the activation of adenylate cyclase [91]. This suggests that signal transduction from the CaR occurs via both pertussis toxin dependent and independent mechanisms depending on the agonist, cellular process and cell type [92].
Fig. 2. Schematic illustration of molecular signalling from the Ca2+ sensing receptor emphasizing PKC as a target down-stream of the receptor
On the cell surface, the CaR exists as a dimer [72, 93-95] and the binding of its agonists to the ECD is highly co-operative [96-99]. Agonist-induced activation of the CaR leads to a conformational change that activates
13 heterotrimeric G proteins. The activation of the latter involves GTP exchange in the GD subunit and the dissociation of the GTP-bound GD subunit from the GEJ subunits. The GD and the GEJ subunits then influence the activities of a number of effector molecules to elicit changes in the cellular levels of second messengers such as cAMP, inositol triphosphates (IP3) and diacylglycerol (DAG) [87, 88, 100-103]. Among these effectors are two isoforms of phospholipase C-E (PLC-E), phosphatidylinositol-dependent PLC-E [104] and phosphatidylcholine- dependent PLC-E [105]; the small GTPase Rho [90, 102]; adenylate cyclase [106, 107]; Ca2+ channels [108, 109] and K+ channels [110]. Downstream of 2+ these effectors, the PLC-IP3- Ca and PLC-DAG-PKC pathways may directly or indirectly influence diverse cellular processes triggered by CaR activation. Other pathways, which may not be exclusive from the above, include the MAP kinase ERK1/2 pathway [111, 112], required for the proliferation and survival of most cells, the activation of cytosolic phospholipase A2 [101], which is necessary for the generation of arachidonate metabolites, and the activation of ER and plasma membrane cation channels that modulate the 2+ >Ca @i [108]. By mechanisms that are yet to be uncovered, agonists of the CaR have also been shown to activate non-receptor tyrosine kinases in fibroblasts and epithelial cells [113, 114]. In PT cells we found that p56Fyn is the most abundant member of these kinases but its activity was not dependent on 2+ changes in >Ca @o (Sakwe AM and Rask L. unpublished observations).
Pathophysiology of the parathyroid glands A number of disorders associated with perturbation of Ca2+ homeostasis have been characterised with the major consequence being either hypercalcemia or hypocalcemia. These have been shown to result predominantly from abnormal functions of the parathyroid that lead to either over-production of PTH (hyperparathyroidism) or decreased synthesis and secretion of PTH (hypoparathyroidism). Hyperparathyroidism (HPT) is most commonly caused by parathyroid adenoma (primary HPT) or by kidney malfunction causing a compensatory over-activity of the parathyroid glands (secondary HPT). In 2+ recent years changes in >Ca @o and/or secreted PTH have also been attributed to mutations that cause either a gain or loss of function of the CaR [73, 115]. The modulation of CaR activity is, thus, believed to be the cause for three inheritable disorders of calcium homeostasis including familial hypocalciuric hypercalcemia (FHH), neonatal severe hyperparathyroidism (NSHPT) and autosomal dominant hypocalcemia with hypercalciuria (ADH) [116-120]. FHH is a benign condition with hypercalcemia that results from heterozygous loss of function mutations of the CaR gene while the homozygous disorder NSHPT is a rare condition with extreme hypercalcemia, which occurs at infancy [121, 122]. ADH, which may be asymptomatic is due to gain of function mutations in the CaR gene [123].
14 Protein kinase C Discovery, structure and classification Protein kinase C (PKC) was originally discovered some 20 years ago as a phospholipid and Ca2+-dependent serine/threonine protein kinase [124]. It is currently known to be a family of enzymes with at least 11 members classified as conventional, novel and atypical PKC on the basis of their structure and co-factor requirements [125, 126]. PKC is activated by cell- surface signal-mediated hydrolysis of inositol phospholipids and transduces EC signals from a variety of receptors to influence several cellular processes [127]. Structurally, the enzymes comprise a regulatory domain in the N- terminal and a catalytic domain in the C-terminal separated by a hinge region at the third variable region. Proteolytic cleavage at the hinge region by trypsin or Ca2+-activated proteases (calpains) generates the regulatory fragment and the constitutively active catalytic fragment [128, 129] currently denoted as PKM. However, depletion of activated PKC is now known to occur by proteasome-mediated degradation [130]. While the catalytic domain is fairly conserved, the response of PKC isoforms to various cofactors is conferred by structural elements in the regulatory domain [126]. The structural features of cPKC-D and nPKC-H used in our studies are shown in Fig. 3.
Fig. 3. Structural features of the Ca2+-dependent PKC-D and the Ca2+-independent PKC-H (adapted from Newton et al., 1995)
15 The regulatory domain of conventional or cPKCs (PKC-D, -EI, -EII and -J) contains two conserved regions C1 and C2. The C1 region of these PKC isoforms is subdivided into two sub-domains denoted as C1A and C1B. Each C1 domain comprises a cysteine-rich zinc finger-like motif, which confers the molecule its ability to bind DAG and phorbol esters, while the C2 region binds Ca2+ and phospholipids [131]. These cPKCs are therefore activated by Ca2+, DAG and phospholipids. The regulatory domain of novel or nPKCs (PKC-G, -H, -K and -T) either lacks or contains a modified C2 domain, while that of atypical or aPKCs (PKC-] and -L/O) lacks the C2 domain, and their C1 domain contains a single cysteine-rich loop. Thus, nPKCs are insensitive to Ca2+ but activated by DAG/phorbol esters and phospholipids, while aPKCs are insensitive to both Ca2+ and DAG/phorbol esters but are activated by phospholipids.
Mechanism of activation and regulation of function Newly synthesized PKC is thought to be an inactive dephosphorylated precursor that is associated with the cytoskeleton [132]. In order for it to attain maturity and become enzymatically active, it is post-translationally modified by three distinct but specific phosphorylations in vivo [133-135]. One of these sites, a conserved Thr residue in the activation loop, is presumably post- or co-translationally phosphorylated by a PKC kinase [136], while the other two are auto-phosphorylation sites at conserved Thr and Ser residues in the C-terminal end of the molecule. Using PKC-EII as a model, Newton and co-workers [135] demonstrated that phosphorylation of PKC in the activation loop (Thr500) by PKC kinase, renders the enzyme catalytically competent that allows it to autophosphorylate first on T641 and then on S660 in
Fig. 4. Translocation of PKC to membranes (adapted from Newton et al., 1995)
16 the C-terminus. Following the series of phosphorylations, the enzyme is released into the cytoplasm where it can respond to lipid second messengers (Fig. 4). The activity of PKC can be assayed in vitro by their phosphotransferase activity in the presence of Mg2+ and ATP; and in vivo by their translocation to cell membranes (Fig. 4) including plasma membrane, nuclear membranes and microsomal membranes [137, 138]. While Ca2+ plays an important role in the translocation of cPKCs, binding of DAG or phorbol esters provokes the translocation of cPKC and nPKC by a mechanism(s) that remain unclear. Translocation of PKC in some cells has been shown to depend on filamentous actin and/or microtubules but is independent of its kinase activity [139].
Assessment of the involvement of PKC in PTH secretion In most cell types including PT cells [140, 141], several PKC isoforms are co-expressed and these are independently affected by extracellular signals. Because PKC isoforms are activated in response to extracellular stimuli, regulate ion channel activity and mediate transmembrane signalling from cell surface receptors, they have been implicated in virtually all cellular processes including secretion [142, 143]. The frequently used procedure to demonstrate the involvement of these enzymes in cellular processes is by interfering with their enzymatic activity. This is carried out by treatment of cells with phorbol esters or DAG analogues that potently activate the at least eight conventional and novel isoforms or by use of PKC-selective inhibitors such as staurosporine, calphostin C, bisindolylmaleimides and many others, which also in most part are not specific. The concomitant use of PKC activators and inhibitors has always been taken as proof of the involvement of the enzyme in a given process. However, interpretations of findings from such studies are not only difficult but also ambiguous for several reasons. Firstly, activation of PKC by treatment of cells with TPA or DAG analogues is not physiological as it by-passes cell surface receptors including the CaR. Secondly, most PKC activity assays are performed in the presence of co- factors and are based on the presence of activation-competent enzyme [135]. The enzyme activities, thus, reflect the total amount of activation-competent enzyme rather than the activation state of the enzyme in the cell. Thirdly, in most early reports the enzyme was considered to be a single molecule rather than a family of at least 11 related enzymes with different co-factor requirement and subcellular distribution. Finally, there is virtually no single protein or peptide substrate identified today which is an efficient substrate for all PKC isoforms. The use of a given peptide or protein substrate implies different maximal velocities for individual isoforms at given starting conditions and, thus, different phosphotransferase activities (Fig. 5).
17 Fig. 5. Schematic illustration of the Phosphotransferase activity of PKC
A more reliable but also less informative assessment of activation of these enzymes is their translocation to particulate fractions of the cells (Fig. 4). This has been refined for live imaging with the use of fluorescent-tagged full-length PKC or fragments of the enzyme [144-146]. In addition, several in vivo experiments have been carried out by over-expressing the catalytically active fragments [147-149], or dominant negative mutants generated by mutation of a key lysine residue in the ATP binding pocket that leads to decreased ATP transferase activity compared to the wild type enzyme [150-153]. However, the validity of such assays also depends on the mechanism of activation of the enzymes, i.e. by physiological or pharmacological stimulus. Previously, the use of TPA and/or PKC inhibitors was generally considered to be sufficient to demonstrate the involvement of PKC in cellular processes. This is the case for the secretion of PTH in PT cells in 2+ which treatment of PT cells with TPA uncoupled the high >Ca @o induced inhibition of PTH secretion and this was reversed by PKC inhibition [8-12, 154]. However, this mode of PKC activation does not indicate whether physiological activation of the enzyme would lead to similar consequences for the cellular process. Furthermore, the belief that PKC isoforms are the major targets of DAG and phorbol esters has in recent years been strongly challenged with evidence that other non-PKC molecules also are targets of these compounds. These molecules have in common with PKC, the C1 domain and include chimaerins (a family of Rac GTPase activating proteins), RasGRPs (exchange factors for Ras/Rap1), DAG kinase and Munc13 isoforms (scaffolding proteins involved in exocytosis) [155-157]. Given that some of these non-PKC targets of TPA/DAG also influence secretion, and that PKC inhibitors are selective but not specific, the role of PKC in CaR-mediated processes including secretion cannot be reliably assessed by pharmacological modulation of its activity.
18 Regulation of Parathyroid Hormone Secretion
2+ In PT cells, the two major events in the >Ca @o-regulated secretion of PTH 2+ include the sensing of changes in >Ca @o by the CaR and the release of PTH 2+ from secretory vesicles (exocytosis) in response to changes in >Ca @o.
Stimulus-secretion coupling Activation of various cell types by certain EC stimuli leads to stimulated release of the contents of secretory vesicles and dense core granules. This comprises an initial phase that is fast, Ca2+ dependent and ATP-independent, and a slower second phase that is Ca2+-independent but ATP-dependent [158]. In general, compounds that elicit activation of cell-surface receptors provoke the hydrolysis of membrane lipids to generate bioactive second 2+ messengers like IP3, which elicits the release of Ca from internal stores. Therefore, in most secretory cells the primary signal for exocytosis is an increase in the concentration of intracellular Ca2+ that results from release of Ca2+ from internal stores [159]. Depending on the strength and duration of the stimulus, release of Ca2+ from internal stores may deplete the stores. The depletion of internal Ca2+ stores as such stimulates Ca2+ influx, a process known as store-operated or capacitative Ca2+ entry that is also thought to be the signal for regulated secretion in some cell types [160-163]. Exocytosis can also be stimulated independently of a Ca2+ signal in a number of cell types including epithelial, adrenal chromaffin and pancreatic E cells [164, 165]. Current evidence using GTP analogues suggests that Ca2+-independent stimulation of exocytosis requires activation of heterotrimeric G proteins [166-169]. The activation of either PKA or PKC [164, 170] has also been shown to mediate Ca2+-independent release of cargo from secretory vesicles in some types of cells.
The molecular events during exocytosis The bulk of available information on the mechanisms of stimulus-secretion coupling has been obtained from genetic, biochemical, structural and functional studies on synaptic vesicle exocytosis [171, 172]. This process is similar to secretion of peptide hormones and proteins in non-neuronal
19 secretory cells and comprises several steps each of which is carried out by a set of specific cellular factors [173]. The release of the contents of secretory vesicles to the extracellular space (exocytosis) can occur by constitutive or regulated pathways. In the constitutive pathway, the vesicles fuse with the plasma membrane in the absence of extracellular stimuli, while the regulated pathway is a more specialized process, in which release of vesicle contents occurs in response to extracellular signals. Two types of vesicles containing PTH have been described; secretory granules and storage granules, both of which contribute to the total secreted PTH [45]. Regulated secretion of PTH is now known to be predominantly from the newly synthesised pool [33, 35, 2+ 174]. Although the secretion of PTH is essentially regulated by >Ca @o, resting parathyroid cells secrete a basal level of PTH that is not Ca2+- regulated [33, 35]. It is not clear whether basal secretion is predominantly derived from the storage granular pool. The principal mechanism by which the contents of the secretory vesicles are believed to be released to the extracellular space is according to the SNARE (Soluble N-ethylmaleimide attachment factor receptor) hypothesis [175, 176]. This hypothesis basically holds that secretory vesicle-bound proteins (v-SNAREs) bind to a heterodimer in the target membrane (t- SNAREs) to form a detergent-insoluble ternary complex necessary to effect fusion of the secretory vesicle and plasma membranes during which a fusion pore is formed to facilitate the release of the contents of the secretory vesicle. In neuronal cells, the v-SNAREs include synaptobrevins or vesicle- associated membrane proteins (VAMPs) while the t-SNAREs include SNAP25 and syntaxins. In addition, several other factors are associated with these proteins including the ATPase N-ethylmaleimide-sensitive factor (NSF), synaptotagmins, soluble NSF attachment proteins (SNAPs) and small GTP binding proteins of the Rab class [177]. SNAP25 contains two SNARE motifs which serve as the binding motif for syntaxin in the heterodimer and also for VAMP in the core complex, which is made up of a four-helix bundle [171, 172, 178]. Several isoforms of these proteins have been identified in both neuronal and endocrine cells [179]. The role of synaptotagmins is probably limited to sensing of intracellular Ca2+ for exocytosis [180]. A number of SNARE proteins have been shown to be phosphorylated by kinases including PKC and this alters the interaction of the individual members in the core complex and consequently, the function of the complex [181, 182].
20 SNARE proteins in PT cells Data on the expression of SNARE proteins and whether these are functional in the regulated secretion of PTH in PT cells is lacking. By RT-PCR we have shown that PT cells express VAMP2, SNAP23, syntaxins 2, 3, 5 and 6 and synaptotagmins II, IV, VI and VIII (Sakwe AM and Rask L, unpublished observations). The expression of SNAP23, and VAMP2 at the protein level was verified by western blotting with antibodies against these proteins. That these proteins are functional in PT cells was confirmed by colocalization of PTH and VAMP (Fig. 5)
PTH VAMP2 Merge
Fig. 6. Co-localization of PTH and VAMP2 (confocal images, courtesy of A. Salnikov)
Regulation of PTH secretion by intracellular degradation Besides the clearly defined proteolytic cleavages necessary to yield the mature bioactive hormone, PTH is degraded intracellularly in PT glands and in the target organs. Such intracellular metabolism of mature PTH in PT cells has been suggested to be one of the mechanisms by which the secreted pool of the hormone is regulated [4]. This line of reasoning was deduced from the fact that proteases such as cyteine proteinases (calpains) that degrade PTH are Ca2+-sensitive and act over a wide range of Ca2+ concentrations [183]. Calpains are located in the Golgi network and include micro-calpains activated at micromolar Ca2+ and milli-calpains that are active at millimolar Ca2+. Another class of PTH degrading proteases are cathepsins and in particular cathepsins B and D in lysosomes [5, 184-187]. Proteolysis of PTH already in the secretory/storage granules might occur by fusion of these granules with lysosomes or in the secretory pathway by maturing cathepsins ( ). Together these proteases have been reported to carry out limited proteolysis of the mature bioactive PTH in the late portions of the secretory pathway to generate PTH fragments of known sizes (PTH1-34, PTH 35-84) [186, 187]. The association of these PTH-proteolysing enzymes
21 with PTH secretion was reported some 20 years ago. This was based on the observations that treatment of PT cells with compounds that inhibited the activities of these proteases stimulated the secretion of PTH. It is not clear whether the effects of these proteases on PTH secretion are Ca2+ dependent, since no evidence of their involvement in regulated exocytosis has been documented. It is therefore possible that intracellular proteolysis and/or degradation of PTH might be a house-keeping function in the biosynthesis of PTH.
Mechanisms of action of pharmacologically active agents on PTH secretion Several studies conducted in the last two decades using acutely dispersed PT cells or PT gland slices demonstrated that incubation of these PT specimens with a number of pharmacological compounds could either stimulate or inhibit the secretion of PTH. The mechanism(s) by which these compounds influence the secretion of PTH are varied and range from inhibition of proteolysis of the mature hormone (see above) to the modulation of the function of the CaR and/or the concentration of intracellular second messengers like intracellular Ca2+, phosphoinositides or other lipid mediators. Divalent cations such as Ca2+, Mg2+, Ni2+, Mn2+, Co2+; polyvalent cations including Gd3+and La3+; polyamines like spermine as well as antibiotics like neomycin affect PTH secretion as agonists of the CaR [6, 188, 189]. Phosphate influences PTH secretion in a manner opposite to that of Ca2+ through mechanisms that may be associated with the shift in equilibrium between free Ca2+ and calcium phosphate complexes [190]. Agents that antagonise Ca2+ entry channels such as verapamil, nifedipine, (+) Bay-K- 8644, (+) 202-791 etc. [191, 192] are believed to affect PTH secretion essentially by modifying Ca2+ fluxes across the plasma membrane and 2+ consequently, the >Ca @i. Potent activators of PKC such as the phorbol ester TPA or DAG analogues like 1,2-dioctanoylglycerol as well as broystatin-1 uncouple the 2+ high >Ca @o-induced inhibition of PTH secretion [8, 9]. The implication of PKC in the secretion of PTH was substantiated in studies utilizing TPA, since these effects were reversed by PKC inhibition. However, as discussed above the interpretation of such experiments is uncertain. Furthermore, the 2+ identity of PKC isoforms and the PKC-dependent mechanism(s) in >Ca @o- regulated secretion of PTH remained undefined.
22 Results and Discussion
Objectives and experimental approach The fundamental question addressed in this dissertation is why an increase in 2+ >Ca @o brings about suppression of PTH secretion in PT cells. 2+ Understanding the mechanism by which >Ca @o sensing by the CaR is coupled to PTH release is paramount to unveiling this question. To provide further insight on this issue, the following specific objectives were investigated:
1. To examine the mode and role of degradation of PTH precursors by proteasomes in the maturation and secretion of PTH. 2+ 2. To examine the relationship between changes in >Ca @o, the activation of the CaR and PKC activation with emphasis on the involvement of the PLC-DAG-PKC and the Raf-MEK-ERK1/2 signalling cascades on PTH secretion. 3. To examine the mechanism of Ca2+ entry following the activation of the CaR and investigate the role of PKC in the modulation of intracellular Ca2+. 4. To investigate the role of PKC-D or -H in regulated secretion of PTH in PT cells.
To achieve the above objectives, dispersed PT cells were used for studies that necessitated assay of secreted PTH. In this case, PT glands were isolated from freshly killed cows at the main slaughterhouse in Uppsala and transported to the laboratory in rich medium supplemented with 10 % fetal bovine serum (FBS). PT cells were dispersed from the glands by collagenase digestion and isolated by centrifugation over Percoll gradients. Cells were allowed to resuscitate in complete medium for 2 hours, then serum-starved for 2 to 4 hours before use in experiments. For studies that were devoted to understanding molecular signalling from the CaR, we established HEK293 cells stably transfected with the wild type receptor or Thr888Ala mutant CaR. The role of PKC-D or -H in CaR signalling and PTH secretion were investigated by over-expressing either wt or DN mutants of these enzymes in HEK-CaR cells by stable transfection or in PT cells by infecting dispersed PT cells with tetracycline-inducible recombinant adenoviruses encoding the wt or DN mutant enzymes.
23 Summary of our observations
Paper I Biosynthesis and secretion of parathyroid hormone are sensitive to proteasome inhibitors in dispersed bovine parathyroid cells 2+ Previous studies revealed that acute changes in >Ca @o did not affect the synthesis of PTH precursors, their processing or the translocation of the molecules through the secretory pathway [193]. However, before the cloning and characterization of the CaR [6], intracellular proteolysis of the mature bioactive hormone by Ca2+-senstive cysteine proteinases was suggested to be 2+ the mechanism by which PTH secretion was regulated by >Ca @o [4, 5]. These investigations were limited to the proteolysis of the mature hormone with little if any information of the PTH precursors. Because protein synthesis occurs in the cytoplasm, we reasoned that the ubiquitous multicatalytic protein degradation machinery, the proteasome, might influence the biosynthesis and secretion of mature PTH. This study, thus, examined how limiting concentrations of the two PTH precursors may affect the biosynthesis of the mature bioactive hormone and consequently, the secretion of intact PTH. The primary precursor, preproPTH, is thought to be co-translationally converted to proPTH [194] making it hard to detect under physiological conditions. In order to quantitatively see this species and manipulate the formation of proPTH, the predominant form in the early part of the ER, we metabolically labelled PT cells with >35S@-methionine in the presence or absence of proteasome inhibitors. In this study, we showed that both preproPTH and proPTH are substrates of the proteasome and that the proteasome plays a quality control function on the maturation of PTH by degrading damaged and/or misfolded PTH precursors. This conclusion is based on the following observations: (1) Inhibition of PT cell proteasomes lead to the accumulation of preproPTH and proPTH. This effect was definitely proteasome-mediated because it differed from the accumulation of proPTH in brefeldin A-treated cells or the accumulation of PTH in chloroquine-treated cells; (2) inhibition of proteasome activity inhibited PTH secretion, while relieving the inhibition reversed the effects on PTH secretion; (3) inhibition of PTH secretion in proteasome-inhibitor treated cells resulted from diminished processing of PTH precursors; (4) the expression of ER (GrP78 or BiP) and cytosolic molecular chaperones was enhanced in proteasome-inhibitor treated cells and these chaperones were found to interact with PTH by co-immunoprecipitation. This indicated that the diminished processing of PTH precursors in proteasome-inhibitor treated cells was due to their retarded transit through the secretory pathway. However, neither proteasome activities nor the accumulation of PTH
24 2+ precursors were observed to be modulated by changes in >Ca @o, suggesting that Ca2+-regulated secretion of PTH in PT cells cannot be attributed to intracellular degradation of PTH and/or its precursors.
Rationale for Papers II, II & IV In spite of the discovery of the CaR in 1993 [6], the confirmation of its role in Ca2+ sensing/signalling in PT cells [189] did not in itself answer the 2+ fundamental question on the inverse relationship between >Ca @o and PTH 2+ secretion. Although increase in >Ca @i as the inhibitory signal for PTH secretion [195] was also observed when PT cells were treated with either 2+ thapsigargin or ionomycin [196, 197] the association of >Ca @o-induced 2+ changes in >Ca @i with changes in PTH secretion remains controversial [7, 198]. Likewise, indirect evidence using TPA and or PKC inhibitor-treated cells indicated a role of PKC in PTH secretion that was contradictory to the lately observed physiological activation of the enzyme upon stimulation of 2+ the CaR. The effects of high >Ca @o on the activity of PKC have also not been unambiguously demonstrated, since in vitro PKC assays do not indicate the activation state of the enzyme in the cell. Since PT cells cannot be manipulated using standard molecular biology techniques, the mechanism by 2+ which >Ca @o regulates PTH secretion has remained obscure. Many laboratories resorted to the use of CaR-transfected HEK293 cells to study processes downstream of the receptor. The components identified to belong to signal transduction chain(s) from the CaR in this model may be valid in PT cells and PTH secretion if verified in PT cells. The following three studies were devoted to molecular signalling from the CaR using HEK-CaR and PT cells with emphasis on the role of PKC in the CaR-mediated secretion of PTH.
Paper II Involvement of protein kinase C-alpha and epsilon in extracellular Ca2+ signalling mediated by the calcium sensing receptor 2+ In this study, we examined the connection between high >Ca @o-activated CaR and PKC activation and evaluated this using one prototype member each of the conventional and novel PKC isoforms that require DAG for 2+ activity. We used the high >Ca @o-induced CaR-mediated activation of the mitogen activated protein kinases ERK1 and ERK2 (ERK1/2) [111, 112] as the read out and the translocation of PKC to the membranes as activity assay. We complemented pharmacological modulation of PKC activity with over-expression in HEK-CaR cells of full-length enzymes prone to similar regulatory mechanisms as the endogenous enzymes. This study revealed that
25 2+ specific PKC isoforms, most likely the cPKC-D, partly mediated the >Ca @o signal that leads to the activation of ERK1/2 and that this required phosphorylation of the CaR on Thr888 and Ca2+ influx via non-selective cation channels. This conclusion was supported by the findings that: (1) in both PT and HEK-CaR cells, PKC-D was predominantly cytosolic at low 2+ 2+ >Ca @o while PKC-H was predominantly membrane-associated. High >Ca @o rapidly activated PKC-D while the activation of PKC-H could not be easily detected presumably due to its membrane location; (2) both TPA and high 2+ >Ca @o induced the activation of ERK1/2 and in either case this was partly inhibited by PKC inhibitors. The TPA-induced activation of ERK1/2 was 2+ 2+ Ca - and CaR-independent; (3) high >Ca @o-induced activation of ERK1/2 required the activity of phosphatidylinositol-dependent phospholipase C-E and influx of Ca2+ via Ni2+-sensitive Ca2+ channels; (4) over-expression of wild type PKC-D or -H augmented the high Ca2+-induced activation of ERK1/2 while over-expression of the dominant negative mutants of these 2+ enzymes attenuated high >Ca @o-induced activation of ERK1/2; (5) over- expression of the T888A mutant CaR in HEK293 cells strongly inhibited the 2+ high >Ca @o-induced activation of ERK1/2. In a recent report using human pathologic and normal parathyroid cells, CaR-mediated activation of ERK1/2 was shown to influence secretion of PTH [112] based on inhibition of the upstream MAP kinase kinase (MEK). However, we could neither reproduce these findings in bovine PT cells, nor demonstrate that inhibition of MEK reversed the TPA stimulated secretion at 2+ high >Ca @o, an effect that was efficiently reversed by PKC inhibition. This 2+ suggests that the high >Ca @o-induced activation of ERK1/2 is not necessary for the Ca2+-regulated secretion of PTH.
Paper III Protein kinase C modulates agonist-sensitive release of Ca2+ from internal stores in HEK293 cells over-expressing the calcium sensing receptor 2+ 2+ Activation of the CaR by high >Ca @o is accompanied by release of Ca 2+ from internal stores and sustained elevation of the >Ca @i, that is believed to be important for the regulation of PTH secretion [195]. The specific objectives of this study were to delineate any role of PKC in the modulation 2+ of >Ca @i following either pharmacological activation/inhibition of its activity or physiological activation via the CaR, and to re-examine the mechanism of Ca2+ influx after receptor activation. Pharmacological inhibition of PKC not only increased the sensitivity of the receptor for its agonists but also the amount of neomycin-induced release of Ca2+ from intracellular stores. Activation of PKC by treatment of cells with TPA 2+ decreased the effective >Ca @i and the magnitude of neomycin-induced
26 release of Ca2+ from intracellular pools. Prolonged treatment of cells with TPA effectively down-regulated immuno-reactive PKC-D but had little effects on the expression levels of PKC-H. Down-regulation of PKC activity as such enhanced the neomycin-induced release of Ca2+ from internal stores. On the contrary, over-expression of wt PKC-D or -H augmented while dominant negative mutant PKC-H strongly inhibited the release of Ca2+ from internal stores. Over-expression of DN PKC-D only slightly decreased the release of Ca2+ from internal stores. Attempts to estimate the rate of Ca2+ influx indirectly from the sustained levels or by loading cells with high concentrations of the Ca2+-buffering chelator BAPTA after receptor activation did not give consistent results. However, using 2-APB which is a reliable blocker of capacitative Ca2+ entry [199], we showed that the influx of Ca2+ following activation of the CaR occurs by store-operated mechanisms. The above data demonstrate that although treatment of cells with TPA activates PKC, the effects of such potent activity decreased the effective 2+ concentration of intracellular >Ca @i, which is opposite to the effects of PKC inhibition. This is consistent with the stimulation of PTH secretion in TPA- treated PT cells and the reversal of the TPA effects upon PKC inhibition. The contradictory effects between physiological activation of PKC via the CaR and pharmacological modulation of the activity of the enzyme strongly suggest that the TPA effects on PTH secretion should be interpreted with caution. These results even question whether PKC plays any direct role in the regulation of PTH secretion.
Paper IV Physiological activation of PKC-alpha via the calcium sensing receptor is not required for the Ca2+-regulated secretion of parathyroid hormone in dispersed bovine parathyroid cells 2+ Studies II and III show that PKC is a component of the >Ca @o signalling system downstream of the CaR. However, whether physiological activation 2+ of PKC is part of the signal transduction chain for >Ca @o-regulated secretion of PTH in PT cells remains unclear. Evidence from pharmacological modulation of the activity of PKC does not seem to be consistent with that from physiological activation of the enzyme via the CaR. This prompted the present study in which any direct involvement of physiologically activated 2+ PKC in the >Ca @o-regulated, CaR-mediated secretion of PTH was examined by over-expression of the DAG-responsive PKC-D and -H in PT cells. Since PT cells are not amenable to transfection, we used the adenovirus-mediated gene delivery system under the control of the tetracycline-inducible promoter (Tet-On) to over-express wt or DN PKC-D or -H in these cells. We demonstrated that these proteins were over-expressed in PT cells following
27 2+ induction of protein synthesis with doxycycline. However, high >Ca @o or neomycin-induced activation of the CaR and consequently physiological activation of either wt or DN PKC-D or -H did not influence PTH secretion. In contrast, treatment of the infected cells over-expressing wt PKC-D with 2+ TPA enhanced the uncoupling of high >Ca @o-induced inhibition of PTH secretion while the corresponding dominant negative mutant had little or no effect. Over-expression of PKC-H as either wt or DN mutant neither influenced the CaR agonist-induced inhibition nor the TPA-stimulated secretion of PTH. This study suggests that although the TPA effect was 2+ enhanced in cells over-expressing PKC-D, the coupling of the >Ca @o signal to PTH secretion in PT cells is not dependent on physiological activation of this PKC isoform via the CaR. This emphasizes that interpretation of the role of PKC in CaR-mediated processes including PTH secretion in PT cells, based on pharmacological modulation of its activity should be interpreted with caution.
Discussion PT cells are probably the only cell type in mammals that can synthesize and secrete PTH. Since PTH is the major hormone responsible for the regulation 2+ 2+ of >Ca @o, it is logical that >Ca @o regulates PTH secretion by feed-back mechanisms. Although this to an extent supports the inverse relationship 2+ between >Ca @o and PTH secretion, the regulatory mechanisms may be exerted at several levels from the biosynthesis and maturation of PTH in the secretory pathway to the release of the hormone from secretory vesicles. Most studies on the biosynthesis of PTH were carried out some 20 years ago and current studies have been clearly devoted to the role of the CaR on cellular processes including secretion. Our studies were aimed at re- 2+ examining the previously evoked notions on PTH biosynthesis and >Ca @o- regulated secretion of the hormone and at elucidating the role of PKC, if any, in PTH secretion. The biosynthesis of mature bioactive PTH, like most secreted proteins entails the translocation of preproPTH into the secretory pathway, and the maturation of PTH from this precursor through specific proteolytic cleavages that remove the signal peptide and subsequently the propeptide. This is consistent with the classical mechanism postulated by Blobel and Dobberstein [200] that is common to all secreted and membrane proteins. Previous studies [4, 28] and our observations on PTH biosynthesis revealed that the proteasome activity on PTH precursors, the transit of proPTH through and its processing in the secretory pathway were not influenced by 2+ 2+ acute changes in >Ca @o. It follows therefore that acute extracellular Ca effects on PTH secretion are downstream of the biosynthesis and maturation of the hormone.
28 The immediate level of control following biosynthesis and packaging of the hormone in secretory vesicles or storage granules is the inactivation of bioactive PTH by limited proteolysis and/or degradation. Interestingly, intracellular degradation of the mature hormone was postulated to be a major mechanism that could explain the inverse relationship between PTH 2+ secretion and >Ca @o [5, 45, 201]. This was based on the observations that inhibition of cellular proteolysis increased the secretion of PTH [5] and supported by data on the inhibition of Ca2+-dependent cysteine proteinases that cleave mature PTH in the Golgi stimulated the secretion of the hormone [183, 184]. However, inhibition of Ca2+-independent lysosomal proteases (cathepsins) that also cleave mature PTH presumably in secretory vesicles or storage granules had similar effects [185, 186]. Moreover, cysteine proteinases also cleave several other proteins including PKC and there is no documentary evidence associating the activities of these and other PTH degrading proteases to regulated secretion per se. Together this implies that degradation of PTH precursors or PTH proteolysis may be house-keeping functions necessary to sort translocation competent preproPTH and correctly folded proPTH for conversion to mature bioactive PTH or to maintain appropriate levels of mature PTH in the cells following changes in the secretory response. Following the cloning of the CaR from PT cells in 1993 [6], it has 2+ become clear that the >Ca @o-regulation of PTH secretion is mediated via this cell surface receptor. In spite of the intense research on the properties of 2+ this receptor, the mechanism by which the >Ca @o signal is coupled to PTH secretion has remained elusive. Activation of the CaR by its agonists 2+ including >Ca @o leads to heterotrimeric G protein-mediated activation of phosphatidylinositol-dependent PLC-E. This in turn, leads to the generation of IP3 and DAG, second messengers that respectively stimulate the release of Ca2+ from internal stores and activation of PKC. Therefore, the high 2+ >Ca @o signal from the CaR apart from generating activated GD and GEJ subunits of heterotrimeric G proteins, leads to the PLC-IP3-Ca2+ and PLC- DAG-PKC cascades that individually or in concert may be responsible for 2+ the regulated secretion of PTH in PT cells. Interestingly, changes in >Ca @i 2+ and activation of PKC have both been associated with the >Ca @o-regulated secretion of PTH, but the evidence for the role of PKC remains indirect. Activation of PKC via a cell-surface receptor like the CaR constitutes physiological activation of the DAG-responsive PKC isoforms that participate in many different cellular processes including secretion. In PT cells, activation of the CaR by its agonists and consequently, physiological activation of PKC, correlates with inhibition of PTH secretion [202-204]. PKC can also be activated by treatment of cells with TPA but this potent 2+ activation of PKC uncouples the high >Ca @o-induced inhibition of PTH secretion [8-12, 154].
29 The effects of agonists of the CaR and those resulting from treatment of cells with TPA reveal an ambiguous role of PKC in PTH secretion that needed to be addressed. Since TPA is cell permeable and it is not known to be an agonist to any known cell surface receptor, the role of PKC in the CaR-mediated secretion of PTH cannot be reliably assessed using TPA. TPA treatment of cells therefore by-passes cell surface receptors and causes global activation of the at least eight DAG-responsive PKC isoforms [205]. It is therefore difficult to distinguish direct involvement of a particular PKC isoform in the control of PTH secretion from indirect secondary or tertiary effects resulting from such potent activation. The physiologically relevant procedure would be to examine signal transduction from the CaR in PT cells. Since PT cells cannot be easily transformed in vitro, such studies have been extensively carried out in CaR-transfected HEK293 cells [78, 89, 101, 108, 206, 207]. This model nevertheless cannot be used to assess PTH secretion, requiring confirmation of any observed effects in PT cells [104, 111]. Using this system and PT cells where necessary, we have complemented pharmacological activation/inhibition of PKC with over-expression of full- length DAG-responsive PKC-D or -H, prone to physiological regulation as the endogenous enzymes. Our studies confirmed that the stimulation of PTH 2+ secretion at high >Ca @o resulting from potent activation of PKC by treatment of PT cells with TPA is Ca2+- and CaR-independent and that the 2+ minute effects of this drug at low >Ca @o suggests that the secretory machinery is operating at its maximum rate during such conditions. The stimulation of PTH secretion in TPA-treated PT cells may, thus, be due to direct and potent activation of PKC by TPA which causes a decrease in the 2+ 2+ >Ca @i by stimulating Ca efflux [208, 209]. In contrast to the TPA effects, 2+ inhibition of PTH secretion by high >Ca @o or other CaR agonists is associated with activation of the CaR and hence physiological activation of PKC. Our observations reveal that physiological activation of PKC enhanced the activity of the CaR that was demonstrated by the increase in the release of Ca2+ from internal stores. The accompanying store-operated Ca2+ entry 2+ eventually leads to a sustained increase in the >Ca @i and theoretically, to inhibition of PTH secretion. Although PKC is an effector of the extracellular Ca2+ signal from the 2+ CaR, its downstream effects in the >Ca @o-regulated secretion of PTH remains to be elucidated. From current evidence in other cell types, the role 2+ of PKC might not be limited to the modulation of >Ca @i by enhancing store release and activating store-operated Ca2+ influx [210-212] but could be extended to modulation of exocytosis [164, 181, 182]. However, over- expression of wt and DN PKC-D or -H in PT cells did not indicate any direct involvement of these PKC isoforms in the control of PTH secretion. On the contrary, treatment of PT cells over-expressing wt PKC-D with TPA 2+ enhanced the uncoupling of high >Ca @o-induced inhibition of PTH secretion while over-expression of the DN PKC-D mutant decreased the effect.
30 Together this revealed that the TPA effects may be mediated predominantly via PKC-D but the inhibitory effects of CaR agonists on PTH secretion are not mediated via this PKC isoform PKC. As mentioned above, apart from the clearly discernible effects of PKC activation on the plasma membrane Ca2+ channels, the activation of the enzyme also influences the activity of the CaR [78], but interpretation of the outcome remains unclear. The PKC-dependent phosphorylation of the CaR in TPA-treated cells was suggested to exert a feed-back control on the receptor [79]. Our results do not support this notion given that the effects of TPA may not be specific. Our data instead suggest that the activity of the receptor may be enhanced by PKC phosphorylation possibly causing a conformational change that allows the receptor to efficiently signal to heterotrimeric G proteins. Thus, mutation of a key PKC phosphorylation site on the CaR tail strongly inhibited CaR-mediated influx of Ca2+ at higher 2+ >Ca @o and activation of ERK1/2 but only minimally affected the release of Ca2+ from internal stores. This suggests that PKC mediated phosphorylation of the receptor is a prerequisite for its activity. This is not surprising, as several GPCRs are known to bind calmodulin to their cytosolic tails [213] and that phosphorylation of these receptors by PKC at the calmodulin binding site, prevents the binding of the latter thereby augmenting their activity [214]. This is consistent with a role of PKC in modulating the activity of the CaR but whether this really is true for this receptor is not yet known. 2+ What then is the primary physiological signal for >Ca @o-regulated secretion of PTH? From the studies presented herein, activation of PKC via the CaR if anything, plays an indirect role by influencing the activities of the CaR and plasma membrane Ca2+ channels. Depending on the stimulus, these actions alter Ca2+ fluxes across the plasma membrane and consequently the 2+ 2+ >Ca @i. Therefore, the fundamental and most important signal for >Ca @o- 2+ regulated secretion of PTH is the change in >Ca @i that accompany the 2+ activation of the CaR but how the changes in >Ca @i affect PTH exocytosis per se remains to be elucidated.
31 Acknowledgements
The work presented herein was in most part performed at the Department of Medical Biochemistry and Microbiology but also at the Department of Medical Cell Biology, Uppsala University. As a large department with shared facilities, the successful execution of my role would have been impossible without the input of several personalities: Professor Lars Rask for the commitment, excellent mentorship and responsibility in making available the required funds and reagents at the time these were necessary. Professor Erik Gylfe for the inspiration and introducing me to both the cuvette system and the single cell methods for Ca2+ measurements. Professor Göran Akusjärvi for yet another difficult project on cloning and expression of proteins in viral vectors. I was fascinated by their keen interest, readiness and prompt action whenever this became necessary. I am also deeply indebted to all the academic, administrative and technical staff of the department for the facilities and ready-made help at every moment it was needed. I am also grateful to all my colleagues in Yaounde, Buea and Uppsala, and particularly my co-authors and all of us in the multi-national corridor D11:3 for cooperation and excellent organisation for a more pleasant working environment. I am also thankful to: * Professor Örjan Zetterqvist, Professor Vincent Titanji and Professor Rune Liminga for all the encouragement and advice before this program and through out these years. * The authorities at the Faculty of Science and the Department of Life Sciences, University of Buea, Cameroon for given me the chance to pursue this program. * Malin Åkeblom, Director of the International Program in the Chemical Sciences, and the Gösta Naeslund Minnesfond for the working visit to the University of Buea, Cameroon. * The Swedish Institute, for a guest research fellowship that enabled me to begin these studies.
Finally, I would like to thank my little ones, Jennifer and Linda for understanding why I had to spend so much time away from you throughout these years; and not the least, Joana for all the assistance and inspiring humour at home after the sometimes difficult days.
32 References
1. Moallem, E., Silver, J. & Naveh-Many, T. (1995) Regulation of parathyroid hormone messenger RNA levels by protein kinase A and C in bovine parathyroid cells, J Bone Miner Res. 10, 447-452. 2. Moallem, E., Kilav, R., Silver, J. & Naveh-Many, T. (1998) RNA-Protein binding and post-transcriptional regulation of parathyroid hormone gene expression by calcium and phosphate, J Biol Chem. 273, 5253-5259. 3. Naveh-Many, T. (1999) Post-transcriptional regulation of the parathyroid hormone gene by calcium and phosphate, Curr Opin Nephrol Hypertens. 8, 415- 419. 4. Habener, J. F., Kemper, B. & Potts, J. T., Jr. (1975) Calcium-dependent intracellular degradation of parathyroid hormone: a possible mechanism for the regulation of hormone stores, Endocrinology. 97, 431-441. 5. MacGregor, R. R. & Bansal, D. D. (1989) Inhibitors of cellular proteolysis cause increased secretion from parathyroid cells, Biochem Biophys Res Commun. 160, 1339-1343. 6. Brown, E. M., Gamba, G., Riccardi, D., Lombardi, M., Butters, R., Kifor, O., Sun, A., Hediger, M. A., Lytton, J. & Hebert, S. C. (1993) Cloning and characterization of an extracellular Ca(2+)-sensing receptor from bovine parathyroid, Nature. 366, 575-580. 7. Russell, J., Zhao, W., Christ, G., Ashok, S. & Angeletti, R. H. (1999) Ca2+- induced increases in steady-state concentrations of intracellular calcium are not required for inhibition of parathyroid hormone secretion, Mol Cell Biol Res Commun. 1, 221-226. 8. Brown, E. M., Redgrave, J. & Thatcher, J. (1984) Effect of the phorbol ester TPA on PTH secretion. Evidence for a role for protein kinase C in the control of PTH release, FEBS Lett. 175, 72-75. 9. Muff, R. & Fischer, J. A. (1986) Stimulation of parathyroid hormone secretion by phorbol esters is associated with a decrease of cytosolic calcium, FEBS Lett. 194, 215-218. 10. Membreno, L., Chen, T. H., Woodley, S., Gagucas, R. & Shoback, D. (1989) The effects of protein kinase-C agonists on parathyroid hormone release and intracellular free Ca2+ in bovine parathyroid cells, Endocrinology. 124, 789- 797. 11. Ridefelt, P., Nygren, P., Hellman, P., Larsson, R., Rastad, J., Åkerström, G. & Gylfe, E. (1992) Regulation of parathyroid hormone release in normal and pathological parathyroid cells exposed to modulators of protein kinase C, Acta Endocrinol (Copenh). 126, 505-509. 12. Racke, F. K. & Nemeth, E. F. (1993) Protein kinase C modulates hormone secretion regulated by extracellular polycations in bovine parathyroid cells, J Physiol. 468, 163-716. 13. Ellis, E. A. (1992) Oxford Textbook of Pathology, Oxford University Press, Oxford.
33 14. Åkerström, G., Malmaeus, J. & Bergström, R. (1984) Surgical anatomy of human parathyroid glands, Surgery. 95, 14-21. 15. De Schaepdrijver, L., Simoens, P., Lauwers, H. & De Geest, J. P. (1988) Topography and identification of the parathyroid glands in the calf, Acta Anat (Basel). 133, 156-161. 16. Cinti, S. & Sbarbati, A. (1995) Ultrastructure of human parathyroid cells in health and disease, Microsc Res Tech. 32, 164-79. 17. Narbaitz, R. & Gartke, K. (1975) Fine structure of chick embryonic parathyroid glands cultured on media with different concentrations of calcium, Rev Can Biol. 34, 91-100. 18. Chen, H., Hayakawa, D., Emura, S., Ozawa, Y., Taguchi, H., Yano, R. & Shoumura, S. (2001) Effect of low calcium diet on the ultrastructure of the rat parathyroid gland, Okajimas Folia Anat Jpn. 78, 153-159. 19. Moreira, J. E., Goncalves, R. P. & Acosta, A. H. (1985) Light- and electron microscopic observations on parathyroid glands in different age groups of rats, Gegenbaurs Morphol Jahrb. 131, 869-882. 20. Mayer, H., Breyel, E., Bostock, C. & Schmidtke, J. (1983) Assignment of the human parathyroid hormone gene to chromosome 11, Hum Genet. 64, 283-285. 21. Naylor, S. L., Sakaguchi, A. Y., Szoka, P., Hendy, G. N., Kronenberg, H. M., Rich, A. & Shows, T. B. (1983) Human parathyroid hormone gene (PTH) is on short arm of chromosome 11, Somatic Cell Genet. 9, 609-616. 22. Zabel, B. U., Kronenberg, H. M., Bell, G. I. & Shows, T. B. (1985) Chromosome mapping of genes on the short arm of human chromosome 11: parathyroid hormone gene is at 11p15 together with the genes for insulin, c- Harvey-ras 1, and beta-hemoglobin, Cytogenet Cell Genet. 39, 200-205. 23. Hendy, G. N., Kronenberg, H. M., Potts, J. T., Jr. & Rich, A. (1981) Nucleotide sequence of cloned cDNAs encoding human preproparathyroid hormone, Proc Natl Acad Sci U S A. 78, 7365-7369. 24. Vasicek, T. J., McDevitt, B. E., Freeman, M. W., Fennick, B. J., Hendy, G. N., Potts, J. T., Jr., Rich, A. & Kronenberg, H. M. (1983) Nucleotide sequence of the human parathyroid hormone gene, Proc Natl Acad Sci U S A. 80, 2127- 2131. 25. Weaver, C. A., Gordon, D. F., Kissil, M. S., Mead, D. A. & Kemper, B. (1984) Isolation and complete nucleotide sequence of the gene for bovine parathyroid hormone, Gene. 28, 319-329. 26. Weaver, C. A., Gordon, D. F. & Kemper, B. (1982) Nucleotide sequence of bovine parathyroid hormone messenger RNA, Mol Cell Endocrinol. 28, 411- 424. 27. Kemper, B., Habener, J. F., Rich, A. & Potts, J. T., Jr. (1975) Microtubules and the intracellular conversion of proparathyroid hormone to parathyroid hormone, Endocrinology. 96, 903-912. 28. MacGregor, R. R. & Cohn, D. V. (1978) The intracellular pathway for parathormone biosynthesis and secretion, Clin Orthop, 244-258. 29. Rapoport, T. A. (1986) Protein translocation across and integration into membranes, CRC Crit Rev Biochem. 20, 73-137. 30. Brodsky, J. L. (1998) Translocation of proteins across the endoplasmic reticulum membrane, Int Rev Cytol. 178, 277-328. 31. Hendy, G. N., Bennett, H. P., Gibbs, B. F., Lazure, C., Day, R. & Seidah, N. G. (1995) Proparathyroid hormone is preferentially cleaved to parathyroid hormone by the prohormone convertase furin. A mass spectrometric study, J Biol Chem. 270, 9517-9525.
34 32. Nakayama, K. (1997) Furin: a mammalian subtilisin/Kex2p-like endoprotease involved in processing of a wide variety of precursor proteins, Biochem J. 327, 625-635. 33. MacGregor, R. R., Hamilton, J. W. & Cohn, D. V. (1975) The by-pass of tissue hormone stores during the secretion of newly synthesized parathyroid hormone, Endocrinology. 97, 178-188. 34. Morrissey, J. J. & Cohn, D. V. (1979) Regulation of secretion of parathormone and secretory protein-I from separate intracellular pools by calcium, dibutyryl cyclic AMP, and (1)-isoproterenol, J Cell Biol. 82, 93-102. 35. Chu, L. L., MacGregor, R. R. & Hamilton, J. W. (1983) Effects of isoproterenol and cycloheximide on parathyroid secretion, Mol Cell Endocrinol. 33, 157-168. 36. Habener, J. F. & Potts, J. T., Jr. (1979) Subcellular distributions of parathyroid hormone, hormonal precursors, and parathyroid secretory protein, Endocrinology. 104, 265-275. 37. Bajpai, S. & Hamilton, J. (1990) The isolation and partial characterization of bovine parathyroid secretory granules, Bone Miner. 9, 9-22. 38. Bohler, U., Blind, E., Vogel, G., Hitzler, W., Flentje, D. & Schmidt-Gayk, H. (1989) Two-site immunochemiluminometric assay of intact human parathyrin in serum with use of a tracer peptide purified by reversed-phase high-performance liquid chromatography, Clin Chem. 35, 215-222. 39. Blind, E., Schmidt-Gayk, H., Armbruster, F. P. & Stadler, A. (1987) Measurement of intact human parathyrin by an extracting two-site immunoradiometric assay, Clin Chem. 33, 1376-1381. 40. Francini, G., Nami, R., Gonnelli, S., Groppa, A., Montagnani, M. & Gennari, C. (1982) Radioimmunoassay for the N-terminal, C-terminal and middle region of human parathyroid hormone in bone diseases, Quad Sclavo Diagn. 18, 486-492. 41. Simon, M. & Cuan, J. (1980) C-terminal parathyrin (parathyroid hormone) radioimmunoassay in serum with commercially available reagents, Clin Chem. 26, 1666-1671. 42. Schultz, V. L., Garner, S. C., Lavigne, J. R. & Toverud, S. U. (1994) Determination of bioactive rat parathyroid hormone (PTH) concentrations in vivo and in vitro by a 2-site homologous immunoradiometric assay, Bone Miner. 27, 121-132. 43. Rucinski, B., Mann, G. N. & Epstein, S. (1995) A new rapid and reproducible homologous immunoradiometric assay for amino-terminal parathyroid hormone in the rat, Calcif Tissue Int. 56, 83-87. 44. Hurwitz, S. (1996) Homeostatic control of plasma calcium concentration, Crit Rev Biochem Mol Biol. 31, 41-100. 45. Cohn, D. V., Kumarasamy, R. & Ramp, W. K. (1986) Intracellular processing and secretion of parathyroid gland proteins, Vitam Horm. 43, 283-316. 46. Brown, E. M. (1993) Mechanisms underlying the regulation of parathyroid hormone secretion in vivo and in vitro, Curr Opin Nephrol Hypertens. 2, 541- 551. 47. Watson, P. H. & Hanley, D. A. (1993) Parathyroid hormone: regulation of synthesis and secretion, Clin Invest Med. 16, 58-77. 48. Wolf, F. & Scarpa, A. (1987) Calcium binding by parathyroid cell plasma membranes, Cell Calcium. 8, 171-183. 49. Brown, E., Enyedi, P., LeBoff, M., Rotberg, J., Preston, J. & Chen, C. (1987) High extracellular Ca2+ and Mg2+ stimulate accumulation of inositol phosphates in bovine parathyroid cells, FEBS Lett. 218, 113-118.
35 50. Shoback, D. M., Membreno, L. A. & McGhee, J. G. (1988) High calcium and other divalent cations increase inositol trisphosphate in bovine parathyroid cells, Endocrinology. 123, 382-389. 51. Kifor, O. & Brown, E. M. (1988) Relationship between diacylglycerol levels and extracellular Ca2+ in dispersed bovine parathyroid cells, Endocrinology. 123, 2723-2729. 52. Gylfe, E., Larsson, R., Johansson, H., Nygren, P., Rastad, J., Wallfelt, C. & Åkerström, G. (1986) Calcium-activated calcium permeability in parathyroid cells, FEBS Lett. 205, 132-136. 53. Nygren, P., Gylfe, E., Larsson, R., Johansson, H., Juhlin, C., Klareskog, L., Åkerström, G. & Rastad, J. (1988) Modulation of the Ca2+-sensing function of parathyroid cells in vitro and in hyperparathyroidism, Biochim Biophys Acta. 968, 253-260. 54. Aida, K., Koishi, S., Tawata, M. & Onaya, T. (1995) Molecular cloning of a putative Ca(2+)-sensing receptor cDNA from human kidney, Biochem Biophys Res Commun. 214, 524-529. 55. Cheng, I., Klingensmith, M. E., Chattopadhyay, N., Kifor, O., Butters, R. R., Soybel, D. I. & Brown, E. M. (1998) Identification and localization of the extracellular calcium-sensing receptor in human breast, J Clin Endocrinol Metab. 83, 703-707. 56. House, M. G., Kohlmeier, L., Chattopadhyay, N., Kifor, O., Yamaguchi, T., Leboff, M. S., Glowacki, J. & Brown, E. M. (1997) Expression of an extracellular calcium-sensing receptor in human and mouse bone marrow cells, J Bone Miner Res. 12, 1959-1970. 57. Romoli, R., Lania, A., Mantovani, G., Corbetta, S., Persani, L. & Spada, A. (1999) Expression of calcium-sensing receptor and characterization of intracellular signaling in human pituitary adenomas, J Clin Endocrinol Metab. 84, 2848-2853. 58. Rasschaert, J. & Malaisse, W. J. (1999) Expression of the calcium-sensing receptor in pancreatic islet B-cells, Biochem Biophys Res Commun. 264, 615- 618. 59. Racz, G. Z., Kittel, A., Riccardi, D., Case, R. M., Elliott, A. C. & Varga, G. (2002) Extracellular calcium sensing receptor in human pancreatic cells, Gut. 51, 705-711. 60. Wonneberger, K., Scofield, M. A. & Wangemann, P. (2000) Evidence for a calcium-sensing receptor in the vascular smooth muscle cells of the spiral modiolar artery, J Membr Biol. 175, 203-212. 61. Cheng, S. X., Okuda, M., Hall, A. E., Geibel, J. P. & Hebert, S. C. (2002) Expression of calcium-sensing receptor in rat colonic epithelium: evidence for modulation of fluid secretion, Am J Physiol Gastrointest Liver Physiol. 283, G240-250. 62. Wang, R., Xu, C., Zhao, W., Zhang, J., Cao, K., Yang, B. & Wu, L. (2003) Calcium and polyamine regulated calcium-sensing receptors in cardiac tissues, Eur J Biochem. 270, 2680-2688. 63. Ruat, M., Molliver, M. E., Snowman, A. M. & Snyder, S. H. (1995) Calcium sensing receptor: molecular cloning in rat and localization to nerve terminals, Proc Natl Acad Sci U S A. 92, 3161-3165. 64. Riccardi, D., Lee, W. S., Lee, K., Segre, G. V., Brown, E. M. & Hebert, S. C. (1996) Localization of the extracellular Ca(2+)-sensing receptor and PTH/PTHrP receptor in rat kidney, Am J Physiol. 271, F951-956. 65. Diaz, R., Hurwitz, S., Chattopadhyay, N., Pines, M., Yang, Y., Kifor, O., Einat, M. S., Butters, R., Hebert, S. C. & Brown, E. M. (1997) Cloning, expression,
36 and tissue localization of the calcium-sensing receptor in chicken (Gallus domesticus), Am J Physiol. 273, R1008-1016. 66. Yarden, N., Lavelin, I., Genina, O., Hurwitz, S., Diaz, R., Brown, E. M. & Pines, M. (2000) Expression of calcium-sensing receptor gene by avian parathyroid gland in vivo: relationship to plasma calcium, Gen Comp Endocrinol. 117, 173-181. 67. Radman, D. P., McCudden, C., James, K., Nemeth, E. M. & Wagner, G. F. (2002) Evidence for calcium-sensing receptor mediated stanniocalcin secretion in fish, Mol Cell Endocrinol. 186, 111-119. 68. Cima, R. R., Cheng, I., Klingensmith, M. E., Chattopadhyay, N., Kifor, O., Hebert, S. C., Brown, E. M. & Soybel, D. I. (1997) Identification and functional assay of an extracellular calcium-sensing receptor in Necturus gastric mucosa, Am J Physiol. 273, G1051-1060. 69. Riccardi, D. (1999) Cell surface, Ca2+(cation)-sensing receptor(s): one or many?, Cell Calcium. 26, 77-83. 70. Janicic, N., Soliman, E., Pausova, Z., Seldin, M. F., Riviere, M., Szpirer, J., Szpirer, C. & Hendy, G. N. (1995) Mapping of the calcium-sensing receptor gene (CASR) to human chromosome 3q13.3-21 by fluorescence in situ hybridization, and localization to rat chromosome 11 and mouse chromosome 16, Mamm Genome. 6, 798-801. 71. Hammerland, L. G., Krapcho, K. J., Garrett, J. E., Alasti, N., Hung, B. C., Simin, R. T., Levinthal, C., Nemeth, E. F. & Fuller, F. H. (1999) Domains determining ligand specificity for Ca2+ receptors, Mol Pharmacol. 55, 642-648. 72. Bai, M., Trivedi, S. & Brown, E. M. (1998) Dimerization of the extracellular calcium-sensing receptor (CaR) on the cell surface of CaR-transfected HEK293 cells, J Biol Chem. 273, 23605-23610. 73. Bai, M., Quinn, S., Trivedi, S., Kifor, O., Pearce, S. H., Pollak, M. R., Krapcho, K., Hebert, S. C. & Brown, E. M. (1996) Expression and characterization of inactivating and activating mutations in the human Ca2+o-sensing receptor, J Biol Chem. 271, 19537-19545. 74. Oda, Y., Tu, C. L., Pillai, S. & Bikle, D. D. (1998) The calcium sensing receptor and its alternatively spliced form in keratinocyte differentiation, J Biol Chem. 273, 23344-23352. 75. Tu, C. L., Chang, W. & Bikle, D. D. (2001) The extracellular calcium-sensing receptor is required for calcium-induced differentiation in human keratinocytes, J Biol Chem. 276, 41079-41085. 76. Awata, H., Huang, C., Handlogten, M. E. & Miller, R. T. (2001) Interaction of the calcium-sensing receptor and filamin, a potential scaffolding protein, J Biol Chem. 276, 34871-3479. 77. Hjälm, G., MacLeod, R. J., Kifor, O., Chattopadhyay, N. & Brown, E. M. (2001) Filamin-A binds to the carboxyl-terminal tail of the calcium-sensing receptor, an interaction that participates in CaR-mediated activation of mitogen- activated protein kinase, J Biol Chem. 276, 34880-34887. 78. Bai, M., Trivedi, S., Lane, C. R., Yang, Y., Quinn, S. J. & Brown, E. M. (1998) Protein kinase C phosphorylation of threonine at position 888 in Ca2+o-sensing receptor (CaR) inhibits coupling to Ca2+ store release, J Biol Chem. 273, 21267-21275. 79. Young, S. H., Wu, S. V. & Rozengurt, E. (2002) Ca2+-stimulated Ca2+ oscillations produced by the Ca2+-sensing receptor require negative feedback by protein kinase C, J Biol Chem. 277, 46871-46876.
37 80. Handlogten, M. E., Shiraishi, N., Awata, H., Huang, C. & Miller, R. T. (2000) Extracellular Ca(2+)-sensing receptor is a promiscuous divalent cation sensor that responds to lead, Am J Physiol Renal Physiol. 279, F1083-1091. 81. Quinn, S. J., Ye, C. P., Diaz, R., Kifor, O., Bai, M., Vassilev, P. & Brown, E. (1997) The Ca2+-sensing receptor: a target for polyamines, Am J Physiol. 273, C1315-1323. 82. Young, S. H. & Rozengurt, E. (2002) Amino acids and Ca2+ stimulate different patterns of Ca2+ oscillations through the Ca2+-sensing receptor, Am J Physiol Cell Physiol. 282, C1414-2142. 83. Conigrave, A. D., Quinn, S. J. & Brown, E. M. (2000) L-amino acid sensing by the extracellular Ca2+-sensing receptor, Proc Natl Acad Sci U S A. 97, 4814- 4819. 84. Mailland, M., Waelchli, R., Ruat, M., Boddeke, H. G. & Seuwen, K. (1997) Stimulation of cell proliferation by calcium and a calcimimetic compound, Endocrinology. 138, 3601-3605. 85. Lin, K. I., Chattopadhyay, N., Bai, M., Alvarez, R., Dang, C. V., Baraban, J. M., Brown, E. M. & Ratan, R. R. (1998) Elevated extracellular calcium can prevent apoptosis via the calcium-sensing receptor, Biochem Biophys Res Commun. 249, 325-331. 86. Brown, E. M. & MacLeod, R. J. (2001) Extracellular calcium sensing and extracellular calcium signaling, Physiol Rev. 81, 239-297. 87. Quitterer, U., Hoffmann, M., Freichel, M. & Lohse, M. J. (2001) Paradoxical block of parathormone secretion is mediated by increased activity of G alpha subunits, J Biol Chem. 276, 6763-6769. 88. Hawes, B. E., van Biesen, T., Koch, W. J., Luttrell, L. M. & Lefkowitz, R. J. (1995) Distinct pathways of Gi- and Gq-mediated mitogen-activated protein kinase activation, J Biol Chem. 270, 17148-17153. 89. Kifor, O., Diaz, R., Butters, R. & Brown, E. M. (1997) The Ca2+-sensing receptor (CaR) activates phospholipases C, A2, and D in bovine parathyroid and CaR-transfected, human embryonic kidney (HEK293) cells, J Bone Miner Res. 12, 715-725. 90. Huang, C., Hujer, K. M., Wu, Z. & Miller, R. T. (2004) The Ca2+-sensing receptor couples to Galpha12/13 to activate phospholipase D in Madin-Darby canine kidney cells, Am J Physiol Cell Physiol. 286, C22-30. 91. Holstein, D. M., Berg, K. A., Leeb-Lundberg, L. M., Olson, M. S. & Saunders, C. (2004) Calcium-sensing receptor-mediated ERK1/2 activation requires Galphai2 coupling and dynamin-independent receptor internalization, J Biol Chem. 279, 10060-10069. 92. Kifor, O., Kifor, I. & Brown, E. M. (2002) Signal transduction in the parathyroid, Curr Opin Nephrol Hypertens. 11, 397-402. 93. Fan, G. F., Ray, K., Zhao, X. M., Goldsmith, P. K. & Spiegel, A. M. (1998) Mutational analysis of the cysteines in the extracellular domain of the human Ca2+ receptor: effects on cell surface expression, dimerization and signal transduction, FEBS Lett. 436, 353-356. 94. Pace, A. J., Gama, L. & Breitwieser, G. E. (1999) Dimerization of the calcium- sensing receptor occurs within the extracellular domain and is eliminated by Cys --> Ser mutations at Cys101 and Cys236, J Biol Chem. 274, 11629-11634. 95. Zhang, Z., Sun, S., Quinn, S. J., Brown, E. M. & Bai, M. (2001) The extracellular calcium-sensing receptor dimerizes through multiple types of intermolecular interactions, J Biol Chem. 276, 5316-5332.
38 96. Brauner-Osborne, H., Jensen, A. A., Sheppard, P. O., O'Hara, P. & Krogsgaard- Larsen, P. (1999) The agonist-binding domain of the calcium-sensing receptor is located at the amino-terminal domain, J Biol Chem. 274, 18382-18386. 97. Hu, J., Hauache, O. & Spiegel, A. M. (2000) Human Ca2+ receptor cysteine- rich domain. Analysis of function of mutant and chimeric receptors, J Biol Chem. 275, 16382-16389. 98. Gama, L. & Breitwieser, G. E. (1998) A carboxyl-terminal domain controls the cooperativity for extracellular Ca2+ activation of the human calcium sensing receptor. A study with receptor-green fluorescent protein fusions, J Biol Chem. 273, 29712-29718. 99. Miedlich, S., Gama, L. & Breitwieser, G. E. (2002) Calcium sensing receptor activation by a calcimimetic suggests a link between cooperativity and intracellular calcium oscillations, J Biol Chem. 277, 49691-49699. 100. Clapham, D. E. & Neer, E. J. (1993) New roles for G-protein beta gamma- dimers in transmembrane signalling, Nature. 365, 403-406. 101. Handlogten, M. E., Huang, C., Shiraishi, N., Awata, H. & Miller, R. T. (2001) The Ca2+-sensing receptor activates cytosolic phospholipase A2 via a Gqalpha - dependent ERK-independent pathway, J Biol Chem. 276, 13941-13948. 102. Pi, M., Spurney, R. F., Tu, Q., Hinson, T. & Quarles, L. D. (2002) Calcium- sensing receptor activation of rho involves filamin and rho-guanine nucleotide exchange factor, Endocrinology. 143, 3830-3838. 103. Liu, K. P., Russo, A. F., Hsiung, S. C., Adlersberg, M., Franke, T. F., Gershon, M. D. & Tamir, H. (2003) Calcium receptor-induced serotonin secretion by parafollicular cells: role of phosphatidylinositol 3-kinase-dependent signal transduction pathways, J Neurosci. 23, 2049-2057. 104. Dare, E., Kifor, O., Brown, E. M. & Weber, G. (1998) Characterization of the phosphatidylinositol-specific phospholipase C isozymes present in the bovine parathyroid and in human kidney HEK293 cells stably transfected with the human parathyroid Ca2+-sensing receptor, J Mol Endocrinol. 21, 7-17. 105. McGehee, D. S., Aldersberg, M., Liu, K. P., Hsuing, S., Heath, M. J. & Tamir, H. (1997) Mechanism of extracellular Ca2+ receptor-stimulated hormone release from sheep thyroid parafollicular cells, J Physiol. 502, 31-44. 106. Chang, W., Chen, T. H., Pratt, S. & Shoback, D. (1998) Regulation of extracellular calcium-activated cation currents by cAMP in parathyroid cells, Am J Physiol. 275, E213-221. 107. Chang, W., Pratt, S., Chen, T. H., Nemeth, E., Huang, Z. & Shoback, D. (1998) Coupling of calcium receptors to inositol phosphate and cyclic AMP generation in mammalian cells and Xenopus laevis oocytes and immunodetection of receptor protein by region-specific antipeptide antisera, J Bone Miner Res. 13, 570-580. 108. Ye, C., Rogers, K., Bai, M., Quinn, S. J., Brown, E. M. & Vassilev, P. M. (1996) Agonists of the Ca(2+)-sensing receptor (CaR) activate nonselective cation channels in HEK293 cells stably transfected with the human CaR, Biochem Biophys Res Commun. 226, 572-579. 109. Yoshida, N., Sato, T., Kobayashi, K. & Okada, Y. (1998) High extracellular Ca2+ and Ca2+-sensing receptor agonists activate nonselective cation conductance in freshly isolated rat osteoclasts, Bone. 22, 495-501. 110. Vassilev, P. M., Kanazirska, M. P., Ye, C., Francis, J., Hong, K. & Brown, E. M. (1997) A flickery block of a K+ channel mediated by extracellular Ca2+ and other agonists of the Ca2+-sensing receptors in dispersed bovine parathyroid cells, Biochem Biophys Res Commun. 230, 616-623.
39 111. Kifor, O., MacLeod, R. J., Diaz, R., Bai, M., Yamaguchi, T., Yao, T., Kifor, I. & Brown, E. M. (2001) Regulation of MAP kinase by calcium-sensing receptor in bovine parathyroid and CaR-transfected HEK293 cells, Am J Physiol Renal Physiol. 280, F291-302. 112. Corbetta, S., Lania, A., Filopanti, M., Vicentini, L., Ballare, E. & Spada, A. (2002) Mitogen-activated protein kinase cascade in human normal and tumoral parathyroid cells, J Clin Endocrinol Metab. 87, 2201-2205. 113. Hobson, S. A., McNeil, S. E., Lee, F. & Rodland, K. D. (2000) Signal transduction mechanisms linking increased extracellular calcium to proliferation in ovarian surface epithelial cells, Exp Cell Res. 258, 1-11. 114. McNeil, S. E., Hobson, S. A., Nipper, V. & Rodland, K. D. (1998) Functional calcium-sensing receptors in rat fibroblasts are required for activation of SRC kinase and mitogen-activated protein kinase in response to extracellular calcium, J Biol Chem. 273, 1114-1120. 115. Mancilla, E. E., De Luca, F. & Baron, J. (1998) Activating mutations of the Ca2+-sensing receptor, Mol Genet Metab. 64, 198-204. 116. Pollak, M. R., Seidman, C. E. & Brown, E. M. (1996) Three inherited disorders of calcium sensing, Medicine (Baltimore). 75, 115-123. 117. Hauache, O. M. (2001) Extracellular calcium-sensing receptor: structural and functional features and association with diseases, Braz J Med Biol Res. 34, 577- 584. 118. Hendy, G. N., D'Souza-Li, L., Yang, B., Canaff, L. & Cole, D. E. (2000) Mutations of the calcium-sensing receptor (CASR) in familial hypocalciuric hypercalcemia, neonatal severe hyperparathyroidism, and autosomal dominant hypocalcemia, Hum Mutat. 16, 281-296. 119. D'Souza-Li, L., Yang, B., Canaff, L., Bai, M., Hanley, D. A., Bastepe, M., Salisbury, S. R., Brown, E. M., Cole, D. E. & Hendy, G. N. (2002) Identification and functional characterization of novel calcium-sensing receptor mutations in familial hypocalciuric hypercalcemia and autosomal dominant hypocalcemia, J Clin Endocrinol Metab. 87, 1309-1318. 120. Brown, E. M. (1997) Mutations in the calcium-sensing receptor and their clinical implications, Horm Res. 48, 199-208. 121. Pollak, M. R., Brown, E. M., Chou, Y. H., Hebert, S. C., Marx, S. J., Steinmann, B., Levi, T., Seidman, C. E. & Seidman, J. G. (1993) Mutations in the human Ca(2+)-sensing receptor gene cause familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism, Cell. 75, 1297-1303. 122. Ho, C., Conner, D. A., Pollak, M. R., Ladd, D. J., Kifor, O., Warren, H. B., Brown, E. M., Seidman, J. G. & Seidman, C. E. (1995) A mouse model of human familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism, Nat Genet. 11, 389-394. 123. Pollak, M. R., Brown, E. M., Estep, H. L., McLaine, P. N., Kifor, O., Park, J., Hebert, S. C., Seidman, C. E. & Seidman, J. G. (1994) Autosomal dominant hypocalcaemia caused by a Ca(2+)-sensing receptor gene mutation, Nat Genet. 8, 303-307. 124. Takai, Y., Kishimoto, A., Iwasa, Y., Kawahara, Y., Mori, T. & Nishizuka, Y. (1979) Calcium-dependent activation of a multifunctional protein kinase by membrane phospholipids, J Biol Chem. 254, 3692-3695. 125. Nishizuka, Y. (1988) The molecular heterogeneity of protein kinase C and its implications for cellular regulation, Nature. 334, 661-665. 126. Newton, A. C. (1995) Protein kinase C: structure, function, and regulation, J Biol Chem. 270, 28495-28498.
40 127. Nishizuka, Y. (1992) Intracellular signaling by hydrolysis of phospholipids and activation of protein kinase C, Science. 258, 607-614. 128. Kishimoto, A., Mikawa, K., Hashimoto, K., Yasuda, I., Tanaka, S., Tominaga, M., Kuroda, T. & Nishizuka, Y. (1989) Limited proteolysis of protein kinase C subspecies by calcium-dependent neutral protease (calpain), J Biol Chem. 264, 4088-4092. 129. Kochs, G., Hummel, R., Fiebich, B., Sarre, T. F., Marme, D. & Hug, H. (1993) Activation of purified human protein kinase C alpha and beta I isoenzymes in vitro by Ca2+, phosphatidylinositol and phosphatidylinositol 4,5-bisphosphate, Biochem J. 291, 627-633. 130. Lee, H. W., Smith, L., Pettit, G. R., Vinitsky, A. & Smith, J. B. (1996) Ubiquitination of protein kinase C-alpha and degradation by the proteasome, J Biol Chem. 271, 20973-20976. 131. Ono, Y., Fujii, T., Igarashi, K., Kuno, T., Tanaka, C., Kikkawa, U. & Nishizuka, Y. (1989) Phorbol ester binding to protein kinase C requires a cysteine-rich zinc-finger-like sequence, Proc Natl Acad Sci U S A. 86, 4868- 4871. 132. Borner, C., Filipuzzi, I., Wartmann, M., Eppenberger, U. & Fabbro, D. (1989) Biosynthesis and posttranslational modifications of protein kinase C in human breast cancer cells, J Biol Chem. 264, 13902-13909. 133. Flint, A. J., Paladini, R. D. & Koshland, D. E., Jr. (1990) Autophosphorylation of protein kinase C at three separated regions of its primary sequence, Science. 249, 408-411. 134. Zhang, J., Wang, L., Schwartz, J., Bond, R. W. & Bishop, W. R. (1994) Phosphorylation of Thr642 is an early event in the processing of newly synthesized protein kinase C beta 1 and is essential for its activation, J Biol Chem. 269, 19578-19584. 135. Keranen, L. M., Dutil, E. M. & Newton, A. C. (1995) Protein kinase C is regulated in vivo by three functionally distinct phosphorylations, Curr Biol. 5, 1394-1403. 136. Pears, C., Stabel, S., Cazaubon, S. & Parker, P. J. (1992) Studies on the phosphorylation of protein kinase C-alpha, Biochem J. 283 ( Pt 2), 515-8. 137. Dekker, L. V. & Parker, P. J. (1994) Protein kinase C--a question of specificity, Trends Biochem Sci. 19, 73-77. 138. Mochly-Rosen, D. (1995) Localization of protein kinases by anchoring proteins: a theme in signal transduction, Science. 268, 247-251. 139. Keenan, C. & Kelleher, D. (1998) Protein kinase C and the cytoskeleton, Cell Signal. 10, 225-232. 140. Shivji, F., Cheng, H., Zwiers, H., Hollenberg, M. D. & Hanley, D. A. (1996) Identification of classical, novel, and atypical protein kinase C isoenzymes in the bovine parathyroid, Endocrinology. 137, 3777-3783. 141. Onyango, I., Hjälm, G., Larsson, M. & Rask, L. (1999) Effects of extracellular calcium on the subcellular translocation of bovine parathyroid PKC isozymes, Exp Cell Res. 247, 9-16. 142. Nishizuka, Y. (2001) The protein kinase C family and lipid mediators for transmembrane signaling and cell regulation, Alcohol Clin Exp Res. 25, 3S-7S. 143. Dempsey, E. C., Newton, A. C., Mochly-Rosen, D., Fields, A. P., Reyland, M. E., Insel, P. A. & Messing, R. O. (2000) Protein kinase C isozymes and the regulation of diverse cell responses, Am J Physiol Lung Cell Mol Physiol. 279, L429-438. 144. Oancea, E., Teruel, M. N., Quest, A. F. & Meyer, T. (1998) Green fluorescent protein (GFP)-tagged cysteine-rich domains from protein kinase C as
41 fluorescent indicators for diacylglycerol signaling in living cells, J Cell Biol. 140, 485-498. 145. Robia, S. L., Ghanta, J., Robu, V. G. & Walker, J. W. (2001) Localization and kinetics of protein kinase C-epsilon anchoring in cardiac myocytes, Biophys J. 80, 2140-2151. 146. Wang, Q. J., Lu, G., Schlapkohl, W. A., Goerke, A., Larsson, C., Mischak, H., Blumberg, P. M. & Mushinski, J. F. (2004) The V5 domain of protein kinase C plays a critical role in determining the isoform-specific localization, translocation, and biological function of protein kinase C-delta and -epsilon, Mol Cancer Res. 2, 129-140. 147. Akita, Y., Ohno, S., Yajima, Y., Konno, Y., Saido, T. C., Mizuno, K., Chida, K., Osada, S., Kuroki, T., Kawashima, S. & et al. (1994) Overproduction of a Ca(2+)-independent protein kinase C isozyme, nPKC epsilon, increases the secretion of prolactin from thyrotropin-releasing hormone-stimulated rat pituitary GH4C1 cells, J Biol Chem. 269, 4653-4660. 148. Lehel, C., Olah, Z., Jakab, G. & Anderson, W. B. (1995) Protein kinase C epsilon is localized to the Golgi via its zinc-finger domain and modulates Golgi function, Proc Natl Acad Sci U S A. 92, 1406-1410. 149. Miller, B. W., Baier, L. D. & Morrison, A. R. (1997) Overexpression of protein kinase C-zeta isoform increases cyclooxygenase-2 and inducible nitric oxide synthase, Am J Physiol. 273, C130-136. 150. Hug, H. & Sarre, T. F. (1993) Protein kinase C isoenzymes: divergence in signal transduction?, Biochem J. 291 ( Pt 2), 329-343. 151. Brodie, C., Bogi, K., Acs, P., Lazarovici, P., Petrovics, G., Anderson, W. B. & Blumberg, P. M. (1999) Protein kinase C-epsilon plays a role in neurite outgrowth in response to epidermal growth factor and nerve growth factor in PC12 cells, Cell Growth Differ. 10, 183-191. 152. St-Denis, A., Chano, F., Tremblay, P., St-Pierre, Y. & Descoteaux, A. (1998) Protein kinase C-alpha modulates lipopolysaccharide-induced functions in a murine macrophage cell line, J Biol Chem. 273, 32787-32792. 153. Soh, J. W., Lee, Y. S. & Weinstein, I. B. (2003) Effects of regulatory domains of specific isoforms of protein kinase C on growth control and apoptosis in MCF-7 breast cancer cells, J Exp Ther Oncol. 3, 115-126. 154. Clarke, B. L., Hassager, C. & Fitzpatrick, L. A. (1993) Regulation of parathyroid hormone release by protein kinase-C is dependent on extracellular calcium in bovine parathyroid cells, Endocrinology. 132, 1168-1175. 155. Kazanietz, M. G. (2002) Novel "nonkinase" phorbol ester receptors: the C1 domain connection, Mol Pharmacol. 61, 759-767. 156. Brose, N. & Rosenmund, C. (2002) Move over protein kinase C, you've got company: alternative cellular effectors of diacylglycerol and phorbol esters, J Cell Sci. 115, 4399-4411. 157. Rhee, J. S., Betz, A., Pyott, S., Reim, K., Varoqueaux, F., Augustin, I., Hesse, D., Sudhof, T. C., Takahashi, M., Rosenmund, C. & Brose, N. (2002) Beta phorbol ester- and diacylglycerol-induced augmentation of transmitter release is mediated by Munc13s and not by PKCs, Cell. 108, 121-133. 158. Theander, S., Lew, D. P. & Nusse, O. (2002) Granule-specific ATP requirements for Ca2+-induced exocytosis in human neutrophils. Evidence for substantial ATP-independent release, J Cell Sci. 115, 2975-2983. 159. Tse, F. W. & Tse, A. (1999) Regulation of exocytosis via release of Ca(2+) from intracellular stores, Bioessays. 21, 861-865.
42 160. Melvin, J. E., Koek, L. & Zhang, G. H. (1991) A capacitative Ca2+ influx is required for sustained fluid secretion in sublingual mucous acini, Am J Physiol. 261, G1043-1050. 161. Powis, D. A., Clark, C. L. & O'Brien, K. J. (1996) Depleted internal store- activated Ca2+ entry can trigger neurotransmitter release in bovine chromaffin cells, Neurosci Lett. 204, 165-168. 162. Koizumi, S. & Inoue, K. (1998) Functional coupling of secretion and capacitative calcium entry in PC12 cells, Biochem Biophys Res Commun. 247, 293-298. 163. Fomina, A. F. & Nowycky, M. C. (1999) A current activated on depletion o intracellular Ca2+ stores can regulate exocytosis in adrenal chromaffin cells, J Neurosci. 19, 3711-3722. 164. Hille, B., Billiard, J., Babcock, D. F., Nguyen, T. & Koh, D. S. (1999) Stimulation of exocytosis without a calcium signal, J Physiol. 520, 23-31. 165. Tse, F. W. & Tse, A. (2000) Stimulation of Ca(2+)-independent exocytosis in rat pituitary gonadotrophs by G-protein, J Physiol. 526, 99-108. 166. Lang, J., Ushkaryov, Y., Grasso, A. & Wollheim, C. B. (1998) Ca2+- independent insulin exocytosis induced by alpha-latrotoxin requires latrophilin, a G protein-coupled receptor, Embo J. 17, 648-657. 167. Padfield, P. J. & Panesar, N. (1998) The two phases of regulated exocytosis in permeabilized pancreatic acini are modulated differently by heterotrimeric G- proteins, Biochem Biophys Res Commun. 245, 332-336. 168. Wollheim, C. B., Ullrich, S., Meda, P. & Vallar, L. (1987) Regulation of exocytosis in electrically permeabilized insulin-secreting cells. Evidence for Ca2+ dependent and independent secretion, Biosci Rep. 7, 443-454. 169. Morgan, A. & Burgoyne, R. D. (1990) Stimulation of Ca2(+)-independent catecholamine secretion from digitonin-permeabilized bovine adrenal chromaffin cells by guanine nucleotide analogues. Relationship to arachidonate release, Biochem J. 269, 521-526. 170. Billiard, J., Koh, D. S., Babcock, D. F. & Hille, B. (1997) Protein kinase C as a signal for exocytosis, Proc Natl Acad Sci U S A. 94, 12192-12197. 171. Lin, R. C. & Scheller, R. H. (1997) Structural organization of the synaptic exocytosis core complex, Neuron. 19, 1087-1094. 172. Chen, Y. A., Scales, S. J. & Scheller, R. H. (2001) Sequential SNARE assembly underlies priming and triggering of exocytosis, Neuron. 30, 161-170. 173. Gerber, S. H. & Sudhof, T. C. (2002) Molecular determinants of regulated exocytosis, Diabetes. 51 Suppl 1, S3-11. 174. Muresan, Z. & MacGregor, R. R. (1994) The release of parathyroid hormone and the exocytosis of a proteoglycan are modulated by extracellular Ca2+ in a similar manner, Mol Biol Cell. 5, 725-737. 175. Sollner, T., Bennett, M. K., Whiteheart, S. W., Scheller, R. H. & Rothman, J. E. (1993) A protein assembly-disassembly pathway in vitro that may correspond to sequential steps of synaptic vesicle docking, activation, and fusion, Cell. 75, 409-418. 176. Gagescu, R. (2000) SNARE hypothesis 2000, Nat Rev Mol Cell Biol. 1, 5. 177. Tooze, S. A., Martens, G. J. & Huttner, W. B. (2001) Secretory granule biogenesis: rafting to the SNARE, Trends Cell Biol. 11, 116-122. 178. Fasshauer, D., Eliason, W. K., Brunger, A. T. & Jahn, R. (1998) Identification of a minimal core of the synaptic SNARE complex sufficient for reversible assembly and disassembly, Biochemistry. 37, 10354-10362. 179. Linial, M. (1997) SNARE proteins--why so many, why so few?, J Neurochem. 69, 1781-1792.
43 180. Koh, T. W. & Bellen, H. J. (2003) Synaptotagmin I, a Ca2+ sensor for neurotransmitter release, Trends Neurosci. 26, 413-422. 181. Polgar, J., Lane, W. S., Chung, S. H., Houng, A. K. & Reed, G. L. (2003) Phosphorylation of SNAP-23 in activated human platelets, J Biol Chem. 278, 44369-44376. 182. Chung, S. H., Polgar, J. & Reed, G. L. (2000) Protein kinase C phosphorylation of syntaxin 4 in thrombin-activated human platelets, J Biol Chem. 275, 25286-25291. 183. Waton, P. H., Mortimer, S. T., Wang, K. K., Croall, D. E. & Hanley, D. A. (1995) Calcium-activated proteases in the bovine parathyroid gland: potential role in degradation of parathyroid hormone to peptide fragments, J Mol Endocrinol. 15, 61-71. 184. Hashizume, Y., Waguri, S., Watanabe, T., Kominami, E. & Uchiyama, Y. (1993) Cysteine proteinases in rat parathyroid cells with special reference to their correlation with parathyroid hormone (PTH) in storage granules, J Histochem Cytochem. 41, 273-282. 185. MacGregor, R. R., Hamilton, J. W., Kent, G. N., Shofstall, R. E. & Cohn, D. V. (1979) The degradation of proparathormone and parathormone by parathyroid and liver cathepsin B, J Biol Chem. 254, 4428-4433. 186. Hamilton, J. W., Jilka, R. L. & MacGregor, R. R. (1983) Cleavage of parathyroid hormone to the 1-34 and 35-84 fragments by cathepsin D-like activity in bovine parathyroid gland extracts, Endocrinology. 113, 285-292. 187. Zull, J. E. & Chuang, J. (1985) Characterization of parathyroid hormone fragments produced by cathepsin D, J Biol Chem. 260, 1608-1613. 188. Ridefelt, P., Hellman, P., Wallfelt, C., Åkerström, G., Rastad, J. & Gylfe, E. (1992) Neomycin interacts with Ca2+ sensing of normal and adenomatous parathyroid cells, Mol Cell Endocrinol. 83, 211-218. 189. Brown, E. M., Pollak, M. & Hebert, S. C. (1995) Molecular mechanisms underlying the sensing of extracellular Ca2+ by parathyroid and kidney cells, Eur J Endocrinol. 132, 523-531. 190. Brown, E. M., Vassilev, P. M., Quinn, S. & Hebert, S. C. (1999) G-protein- coupled, extracellular Ca(2+)-sensing receptor: a versatile regulator of diverse cellular functions, Vitam Horm. 55, 1-71. 191. Pocotte, S. L., Ehrenstein, G. & Fitzpatrick, L. A. (1995) Role of calcium channels in parathyroid hormone secretion, Bone. 16, 365S-372S. 192. Ridefelt, P., Hellman, P., Rastad, J., Larsson, R., Åkerström, G. & Gylfe, E. (1996) Effects of calcium channel modulators on the regulation of cytoplasmic Ca2+ and hormone secretion of parathyroid cells, Pharmacol Toxicol. 78, 147- 153. 193. Kemper, B. (1986) Molecular biology of parathyroid hormone, CRC Crit Rev Biochem. 19, 353-379. 194. Cioffi, J. A., Allen, K. L., Lively, M. O. & Kemper, B. (1989) Parallel effects of signal peptide hydrophobic core modifications on co-translational translocation and post-translational cleavage by purified signal peptidase, J Biol Chem. 264, 15052-15058. 195. Wallfelt, C., Lindh, E., Larsson, R., Johansson, H., Rastad, J., Åkerström, G. & Gylfe, E. (1988) Kinetic evidence for cytoplasmic calcium as an inhibitory messenger in parathyroid hormone release, Biochim Biophys Acta. 969, 257- 262. 196. Shoback, D. M., Thatcher, J., Leombruno, R. & Brown, E. M. (1984) Relationship between parathyroid hormone secretion and cytosolic calcium
44 concentration in dispersed bovine parathyroid cells, Proc Natl Acad Sci U S A. 81, 3113-3117. 197. Ferzandi, T. R. & MacGregor, R. R. (1997) Thapsigargin shifts the Ca set point of parathyroid cells to lower extracellular [Ca], Endocrine. 7, 361-366. 198. Nemeth, E. F., Wallace, J. & Scarpa, A. (1986) Stimulus-secretion coupling in bovine parathyroid cells. Dissociation between secretion and net changes in cytosolic Ca2+, J Biol Chem. 261, 2668-2674. 199. Bootman, M. D., Collins, T. J., Mackenzie, L., Roderick, H. L., Berridge, M. J. & Peppiatt, C. M. (2002) 2-aminoethoxydiphenyl borate (2-APB) is a reliable blocker of store-operated Ca2+ entry but an inconsistent inhibitor of InsP3- induced Ca2+ release, Faseb J. 16, 1145-1150. 200. Blobel, G. & Dobberstein, B. (1975) Transfer of proteins across membranes. I. Presence of proteolytically processed and unprocessed nascent immunoglobulin light chains on membrane-bound ribosomes of murine myeloma, J Cell Biol. 67, 835-851. 201. Morrissey, J. J. & Cohn, D. V. (1979) Secretion and degradation of parathormone as a function of intracellular maturation of hormone pools. Modulation by calcium and dibutyryl cyclic AMP, J Cell Biol. 83, 521-528. 202. Åkerström, G., Rastad, J., Ljunghall, S., Ridefelt, P., Juhlin, C. & Gylfe, E. (1991) Cellular physiology and pathophysiology of the parathyroid glands, World J Surg. 15, 672-680. 203. Mithal, A., Kifor, O., Kifor, I., Vassilev, P., Butters, R., Krapcho, K., Simin, R., Fuller, F., Hebert, S. C. & Brown, E. M. (1995) The reduced responsiveness of cultured bovine parathyroid cells to extracellular Ca2+ is associated with marked reduction in the expression of extracellular Ca(2+)-sensing receptor messenger ribonucleic acid and protein, Endocrinology. 136, 3087-3092. 204. Kaneko, C., Mizunashi, K., Tanaka, M., Uzuki, M., Kikuchi, M., Sawai, T. & Goto, M. M. (1999) Relationship between Ca-dependent change of serum PTH and extracellular Ca2+-sensing receptor expression in parathyroid adenoma, Calcif Tissue Int. 64, 271-272. 205. Ryves, W. J., Evans, A. T., Olivier, A. R., Parker, P. J. & Evans, F. J. (1991) Activation of the PKC-isotypes alpha, beta 1, gamma, delta and epsilon by phorbol esters of different biological activities, FEBS Lett. 288, 5-9. 206. Bösel, J., John, M., Freichel, M. & Blind, E. (2003) Signaling of the human calcium-sensing receptor expressed in HEK293-cells is modulated by protein kinases A and C, Exp Clin Endocrinol Diabetes. 111, 21-26. 207. Hobson, S. A., Wright, J., Lee, F., McNeil, S. E., Bilderback, T. & Rodland, K. D. (2003) Activation of the MAP kinase cascade by exogenous calcium- sensing receptor, Mol Cell Endocrinol. 200, 189-198. 208. Gylfe, E. (1989) Phorbol ester desensitization of clonal insulin-releasing cell response to carbachol involves depletion of an intracellular calcium pool, Acta Physiol Scand. 135, 107-111. 209. Kuo, T. H., Wang, K. K., Carlock, L., Diglio, C. & Tsang, W. (1991) Phorbol ester induces both gene expression and phosphorylation of the plasma membrane Ca2+ pump, J Biol Chem. 266, 2520-2525. 210. Putney, J. W., Jr. & McKay, R. R. (1999) Capacitative calcium entry channels, Bioessays. 21, 38-46. 211. Kondo, I. (2000) Protein kinase C potentiates capacitative Ca2+ entry that links to steroidogenesis in bovine adrenocortical cells, Jpn J Pharmacol. 82, 210-217.
45 212. Dyachok, O. & Gylfe, E. (2001) Store-operated influx of Ca(2+) in pancreatic beta-cells exhibits graded dependence on the filling of the endoplasmic reticulum, J Cell Sci. 114, 2179-2186. 213. Wang, D., Sadee, W. & Quillan, J. M. (1999) Calmodulin binding to G protein-coupling domain of opioid receptors, J Biol Chem. 274, 22081-22088. 214. Bofill-Cardona, E., Kudlacek, O., Yang, Q., Ahorn, H., Freissmuth, M. & Nanoff, C. (2000) Binding of calmodulin to the D2-dopamine receptor reduces receptor signaling by arresting the G protein activation switch, J Biol Chem. 275, 32672-32680.
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Acta Universitatis Upsaliensis Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine Editor: The Dean of the Faculty of Medicine
A doctoral dissertation from the Faculty of Medicine, Uppsala University, is usually a summary of a number of papers. A few copies of the complete dissertation are kept at major Swedish research libraries, while the sum- mary alone is distributed internationally through the series Comprehen- sive Summaries of Uppsala Dissertations from the Faculty of Medicine. (Prior to October, 1985, the series was published under the title “Abstracts of Uppsala Dissertations from the Faculty of Medicine”.)
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