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Prepublished online as a Blood First Edition Paper on October 10, 2002; DOI 10.1182/blood-2002-08-2384.
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Department of Biochemistry, University of
Oxford, Oxford, United Kingdom, and the Department of
Pharmacology, University of Oxford, Oxford, United
Kingdom.
Protein kinase D (PKD, also known as PKCµ) is closely related to
the protein kinase C superfamily but is differentially regulated and
has a distinct catalytic domain that shares homology with Ca2+-dependent protein kinases. PKD is highly expressed in
hematopoietic cells and undergoes rapid and sustained activation upon
stimulation of immune receptors. PKD is regulated through
phosphorylation by protein kinase C (PKC). In the present study, we
show that PKD is expressed in human platelets and that it is
rapidly activated by receptors coupled to heterotrimeric G-proteins or
tyrosine kinases. Activation of PKD is mediated downstream of PKC.
Strong agonists such as convulxin, which acts on GPVI, and thrombin
cause sustained activation of PKC and PKD, whereas the thromboxane
mimetic U46619 gives rise to transient activation of PKC and PKD.
Activation of PKD by submaximal concentrations of phospholipase
C-coupled receptor agonists is potentiated by
Gi-coupled receptors (eg, adenosine diphosphate
and epinephrine). This study shows that PKD is rapidly activated by a
wide variety of platelet agonists through a PKC-dependent pathway.
Activation of PKD enables phosphorylation of a distinct set of
substrates to those targeted by PKC in platelets.
(Blood. 2003;101:1392-1399) Protein kinase C (PKC) is a family of
serine/threonine kinases implicated in the signal transduction of a
variety of extracellular stimuli such as hormones and growth
factors.1-4 The extended PKC family involves 11 isozymes
with structural similarities,5-7 which are divided into
subgroups on the basis of structural, enzymatic and regulatory
differences.8 The PKC family members are known to have
distinct mechanisms of activation, patterns of expression, and
subcellular localization in different cell types.8 The conventional PKCs are regulated by Ca2+,
sn-1,2-diacylglycerol (DAG), and phospholipids. The novel PKCs lack a
Ca2+ binding domain but are regulated by DAG and
phospholipids. The atypical PKCs are not known to be regulated by
Ca2+ or DAG.9 All of these subgroups have a
highly conserved catalytic domain and pseudosubstrate
region.3,9,10 PKD (also known as PKCµ) was initially
identified as a member of the PKC family,11 creating a new
subdivision of PKC.
PKD has similarities to other PKCs in that it has a domain that is
homologous to the DAG binding domain of other PKCs, but it lacks the C2
domain responsible for Ca2+ sensitivity of the conventional
PKC subgroup.11,12 In contrast to other PKC isoforms, PKD
has a putative pleckstrin homology (PH) domain that is reminiscent of
those found in the protein kinase B family and is important for
regulating PKD's enzyme activity.13,14 Unlike other PKC
isoforms that have a highly conserved catalytic domain, PKD's
catalytic domain shows a higher degree of homology to the
Ca2+-calmodulin-regulated kinases,13 giving
it a unique substrate specificity.15,16 These differences
and the identification of 2 further PKD isotypes17,18 have
made it difficult to classify PKD. It is currently regarded as a member
of the PKC family by some groups and has recently been classified as a
new novel subgroup of the AGC family of serine/threonine
kinases.19
PKD is highly expressed in hematopoietic cells11,15 and is
rapidly activated through a phosphorylation-dependent
mechanism.20 A variety of stimuli, including phorbol
esters and G protein-coupled receptor and tyrosine kinase-linked
receptor agonists,21 all lead to sustained PKD activation.
Several lines of evidence suggest that this activation is mediated
through a PKC-dependent signal transduction pathway.12,20
Residues Ser744 and Ser748 in the activation loop of PKD have been
shown to be critical sites of phosphorylation that lead to PKD
activation and subsequent autophosphorylation at other residues, such
as Ser916.22,23 The degree of phosphorylation of Ser916
has been used as a measure of PKD activation using phosphospecific antisera.22-26
In platelets, many receptor agonists lead to activation of PKC via the
phosphoinositide second-messenger pathway.27 This involves
activation of phospholipase C (PLC), which cleaves phosphatidyl inositol 4,5-bisphosphate, generating the second messengers inositol 1,4,5-trisphosphate (IP3) and DAG. IP3
mobilizes Ca2+ from intracellular stores and DAG activates
certain isoforms of PKC. Several studies have shown a role for PKC
activation in aggregation and dense granular
secretion28,29 in response to a variety of agonists. This
has been supported by studies that show a correlation between serotonin
release from dense granules and phosphorylation of pleckstrin (a marker
for PKC activation in platelets).30,31 PKC has also been
implicated in platelet adhesion to collagen32 and has been
shown to have a direct role in aggregation that is independent of
secretion from dense granules.33 In spite of PKC's being
implicated in multiple functions during platelet signaling, the role of
individual isoforms is still poorly characterized. Purification studies
and the use of isoform-specific antibodies have shown that platelets
express several isoforms of PKC.34-36 Recent studies in
platelets37 have implicated PKC The objective of the current study was to investigate the regulation of
PKD in human platelets. The results provide the first demonstration
that PKD is present in platelets and that it is activated by G
protein-coupled and tyrosine kinase-linked receptor agonists in these
cells. Unusually, activation of PKD in response to some agonists is
transient, and we show that PKD activation is dependent on sustained
PKC activity. We also demonstrate synergistic activation between
submaximal concentrations of agonists that activate PLC by both G
protein and tyrosine kinase pathways and agonists that stimulate
Gi/z-dependent signaling pathways in platelets.
Materials
Agonists, inhibitors, and reagents.
Adenosine diphosphate (ADP), apyrase, A3P5P (adenosine 3'-phosphate
5'-phosphate), BAPTA-AM (1,2-bis(2-aminophenoxy)ethane-N, N, N, N', N'-tetraacetic acid), epinephrine, fibrinogen, and wortmannin were obtained from Sigma (Poole, United Kingdom). LY294002
(2-[4-morpholoinyl]-8-phenyl-1[4H]-benzopyran-4-one), phorbol-12-myristate-13-acetate (PMA), and U46619
(9,11-dideoxy-11 Antibodies and substrates.
Rabbit antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) were used
for PKD detection. The first (sc639) was raised against a synthetic
peptide with an amino acid sequence corresponding to amino acids
899-918 of murine PKD, and the second (sc937) was raised against a
peptide mapping to 893-912 of the carboxy terminus of human PKD. PA-1
antiserum, pS916 and syntide-2 were gifts from Dr Doreen Cantrell
(Cancer Research UK, London, United Kingdom). The PA-1 antiserum was
raised against the synthetic peptide EEREMKALSERVSIL, a sequence from
the carboxy terminus of PKD.12 PS916 is a phosphospecific antibody directed against phosphoserine 916 of PKD.23
Syntide-2 (PLARTLSVAGLPGKK) is a peptide based on phosphorylation site
2 of glycogen synthase and acts as a substrate for
PKD.12,16,20
Methods
Preparation of platelets.
Blood was drawn on the day of the experiment from aspirin-free
volunteers into acid citrate dextrose (ACD) as anticoagulant. Studies
were carried out with ethical approval from the Central Oxford Research
Committee (Ref: C00:203). Washed platelets were prepared
essentially as described previously by Gear.40 In
brief, platelet-rich plasma (PRP) was obtained by centrifugation at
200g for 20 minutes. Platelets were isolated from PRP by
centrifugation at 450g for 15 minutes in the presence of 5%
(vol/vol) ACD, 5 U/mL apyrase, 80 nM prostacyclin, and 2.8 µM
indomethacin. The pellet was then resuspended in ACD containing 5 U/mL
apyrase and 2.8 µM indomethacin and recentrifuged at 450g
for 15 minutes. Platelets were resuspended to a cell density of
1 × 109/mL in HEPES
(N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid )-buffered
Tyrodes (140 mM NaCl, 5 mM KCl, 15 mM HEPES, 5 mM glucose, 0.4 mM
NaH2PO4, 11.9 mM NaHCO3, and 1 mM
MgCl2). Platelets were incubated for at least 1 hour at
room temperature and just before experimentation 1 mM CaCl2
and 0.2 mg/mL fibrinogen were added. All experiments were performed in
the presence of the cycloxygenase inhibitor indomethacin (10 µM) to
eliminate positive feedback from thromboxanes, with the exception of
studies using the thromboxane mimetic U46619. For synergy experiments,
where epinephrine was used, inhibition of ADP activity was achieved
with the ADP scavenging enzyme apyrase (2 U/mL).
Western blot analysis.
After stimulation, cells were lysed in 2 × Laemmli sample buffer
analyzed by 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene
difluoride (PVDF) membranes (Millipore, Bedford, MA).
Immunodetection was performed with different antibodies. The Santa Cruz
antibodies sc937 and sc639 and the pS916 antisera were used at 1:500
dilution, and the PA-1 antiserum was diluted to 1:100. Immunoreactive
bands were visualized by enhanced chemiluminescence.
Immunoprecipitation and in vitro kinase assays.
After stimulation, cells were lysed at 4°C in lysis buffer (50 mM
Tris/HCl, pH 7.6; 2 mM EGTA [ethyleneglycoltetraacetic acid]; 2 mM EDTA [ethylenediaminetetraacetic acid]; 2 mM
dithiothreitol; protease inhibitors aprotinin (10 µg/mL), leupeptin
(100 µg/mL), and pepstatin (0.7 µg/mL); and 1 mM
4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride, 2 mM sodium
orthovanadate, 1 mM sodium fluoride, 40 mM Phosphatidic acid and pleckstrin phosphorylation.
Phosphatidic acid production was measured as described
previously.41 Washed platelets were labeled with
32P-orthophosphoric acid (0.5 mCi/mL [18.5 × 106 Bq]) for 1 hour at 37°C. After stimulation,
cells were lysed in 2 × Laemmli sample buffer and analyzed by 10%
SDS-PAGE, stained with Coomassie blue, destained, and dried
down. They were then exposed to hyperfilm overnight.
Pleckstrin bands were then excised from the gel and analyzed by
scintillation counting.
Data analysis.
Each experiment was performed at least 3 times. Results are expressed
as the mean ± SEM and were analyzed by an unpaired Student t test with P < .05 taken as the minimum value
to indicate statistical significance.
PKD is expressed in human platelets
PKD is activated in response to PMA in human platelets
PKD is activated in response to the platelet agonists convulxin, thrombin, and U46619 Three platelet agonists were tested for their ability to activate PKD. Convulxin selectively activates the tyrosine kinase-coupled collagen receptor glycoprotein VI (GPVI), and thrombin activates protease-activated receptor (PAR) receptors coupled to heterotrimeric Gq and G12/13 proteins. The thromboxane A2 (TxA2) mimetic, U46619, activates platelets through the Gq- and G12/13-coupled thromboxane prostanoid TP receptor. All 3 of these agonists lead to PKC activation via PLC-mediated generation of DAG.45,46 Convulxin and thrombin stimulated a rapid 4- to 5-fold increase in PKD catalytic activity as assessed by an in vitro kinase assay (Figure 1B). PKD activity reached a maximum after one minute of activation by either agonist and was maintained for 10 minutes. Similarly, Western blot analysis using the pS916 antiserum showed phosphorylation of PKD after 10 seconds, which was sustained for at least 10 minutes for convulxin and thrombin (Figure 1B). These results suggest PKD activation as an early event in convulxin and thrombin signaling during platelet activation. PKD was also activated by U46619, but, in contrast to convulxin and thrombin, activation was shown to be weak and transient (Figure 1B).PKD activation is regulated by a PKC-dependent signal transduction pathway in human platelets In all cells studied so far PKD activation has been shown to be dependent on an active PKC. The next consideration was to assess the role PKC has in the regulation of PKD by receptor stimuli in platelets. Prior to stimulation platelets were preincubated with Ro 31-8220, a potent inhibitor of PKC47 but not PKD.20,21 Preincubating platelets with Ro 31-8220 blocked PKD activation induced by PMA, convulxin, and thrombin (Figure 2). However, addition of the PKC inhibitor directly to PKD during the in vitro kinase assay did not effect the catalytic activity of PKD itself. These results show that receptor activation of PKD is regulated by a PKC-dependent signaling pathway in platelets. These data also show that in spite of each agonist's having a different mechanism of activation, PKD is a common downstream signaling target during platelet activation.
U46619 induces transient activation of PKC Owing to the transient nature of PKD activation induced by U46619, experiments were designed to assess whether prolonged PKC activation is required to maintain PKD activity. Studies were carried out to observe the activity of PKC by measuring pleckstrin phosphorylation. Results show that convulxin-induced PKC activation was increased 5-fold compared with basal activation and was maintained for 5 minutes (Figure 3A). Similar results were observed for thrombin (Figure 4) and PMA (not shown). However, a maximum concentration of U46619 induced a 3-fold increase in pleckstrin phosphorylation (Figure 3A) within 10 seconds of stimulation. This phosphorylation rapidly decreased by approximately 60% after 30 seconds and continued to decrease to less than 10% after 5 minutes. These data show that U46619 is able to transiently activate PKC.
To investigate this further, studies were carried out to observe the activity of PKD upon PKC inhibition. Ro31-8220 was added to platelets 10 seconds after stimulation by PMA, convulxin, or thrombin, and the level of PKD autophosphorylation was measured using the antiphosphoserine antibody p5916. The bands on the resulting autorad were quantified using densitometry and were plotted versus time (Figure 3B). The phosphorylated state of PKD decreased with time for all 3 stimuli, showing that PKC must remain active to maintain PKD activity. These results suggest that PKD activation is regulated by a PKC-dependent signaling pathway and that continued PKC activity is necessary for sustained PKD activation in platelets. It is therefore clear that transient PKD activation in response to U46619 is the result of transient PKC activation induced by this agonist. Other signaling pathways that contribute to PKD activation Other components of the signaling pathway leading to PKD activation by PMA and convulxin were investigated by determining the effects of preincubating platelets with the Src kinase inhibitor PP1, the PI 3-kinase inhibitors LY294002 (not shown) and wortmannin, and the calcium chelator BAPTA-AM (Figure 4). For the phorbol ester, PMA (Figure 4A), there was a small but not significant decrease in PKD activation in the presence of inhibitors of PI3-kinase or Src kinases. There was also approximately 50% inhibition of PKD activation in the presence of the Ca2+ chelator BAPTA-AM, consistent with a role for the cation in the activation of PKC. Convulxin-induced PKD activation (Figure 4B) was inhibited to almost basal levels in the presence of Src kinase inhibition or calcium chelation, whereas PI 3-kinase inhibition decreased PKD activation by more than 60%. This is not surprising, because activation of PLC 2,
by GPVI, is critically dependent on Src kinases48,49 and
is modified in part via PI 3-kinase.39
Thrombin-induced PKD activation was shown to be dependent on PI 3-kinase and Ca2+ with more than 60% attenuation of PKD activation in the presence of wortmannin or BAPTA-AM after 2 minutes (Figure 4 and not shown). A time course of the effects of wortmannin on thrombin-induced PKD activation was compared with thrombin-induced PKC activation (pleckstrin phosphorylation). The results (Figure 4C) show that PKD activation is maintained for 1 minute and is attenuated significantly after 2 minutes, returning to basal levels after 5 minutes. This suggests a role for PI 3-kinase or its products in later stages of PKD regulation. The same pattern of regulation was observed for PKC activation and correlates well with published data50,51 showing a role for PI 3-kinase in the later stages (after 2 minutes) of pleckstrin phosphorylation downstream of the thrombin PAR receptors (Figure 4C). Gi/Gz involvement in activation of PKD Previous work has suggested synergistic activation of platelets by submaximal concentrations of agonists that activate Gq- and Gi/z-dependent signaling pathways.52-56 The pathways activated by Gi/z are not defined but are likely to include PI 3-kinase.39,57 To test this possibility, PKD activation by epinephrine (which activates both Gi- and Gz-linked pathways via the 2A-adrenoceptor) and ADP (which activates both
Gq-[via the P2Y1 receptor] and
Gi-[via the P2Y12 receptor] coupled pathways)
was determined. Epinephrine was unable to activate PKD even at
high concentrations (Figure 5A-Bi).
However, a submaximal concentration of U46619, which was unable to
cause PKD activation alone, stimulates a 4-fold increase in PKD
activation in the presence of epinephrine (Figure 5A-Bi). Similar
experiments carried out with a high concentration of ADP (Figure
5A-Bii) show that activation of Gq and
Gi/z-coupled ADP receptors is required for PKD activation.
In the presence of selective ADP receptor antagonists A3P5P (which
blocks P2Y1) or AR-C67085 (which blocks P2Y12),
there was no activation of PKD. These results reveal that signals from
Gi/z- and Gq-coupled receptors can act together
to activate PKD.
Synergy between GPVI and Gi/z leads to PKD activation Previous work has shown synergy between the tyrosine kinase pathway activated by GPVI and a Gi/z-coupled pathway during platelet aggregation,33,58,59 and so the possibility of synergy between these signaling pathways to activate PKC and thereafter PKD was monitored. A submaximal concentration of convulxin, which on its own stimulated a small increase in PKD activation, was potentiated 4-fold in the presence of ADP (+A3P5P) or epinephrine, as determined by Western blot analysis and in vitro kinase assays (Figure 6). These results demonstrate that concomitant signals from Gi/z-coupled receptors and GPVI can act together to stimulate PKD activation.
Synergistic activation of PKD is at the level of PKC activity The signals from Gi/z that synergize with Gq- or tyrosine kinase (TK)-linked signaling pathways to activate PKD either could be at the level of PKC activation or could represent a separate input directly into PKD activation. To explore these possibilities, PKC activation was assessed by pleckstrin phosphorylation and PLC activity by phosphatidic acid (PA) production. PA is generated in cells by phosphorylation of DAG and so can be used as an indirect measure of DAG levels or PLC activation.60 In both cases a marked potentiation was seen between submaximal concentrations of convulxin or U46619 and epinephrine, at the level of both pleckstrin phosphorylation and phosphatidic acid production (Figure 7). The same results were found when using ADP in the presence of A3P5P, rather than epinephrine, as a selective Gi/z agonist. Thus, activation of Gi/z leads to potentiation of PLC activation by Gq- and TK-linked pathways, which subsequently leads to PKC, then PKD, activation.
Identifying potential kinases activated downstream of platelet receptor stimulation allows a greater understanding of platelet signal transduction. This study implicates PKD as a signaling molecule in the regulation of platelet function. PKD was initially identified as a member of the PKC superfamily but can be distinguished from other PKC isoforms because of differences in its catalytic domain structure, giving it unique substrate specificity.2467 Most important, PKD activation is mediated by phosphorylation by other members of the PKC family, rather than by direct interaction with DAG.62,63 The magnitude and duration of PKD activation by maximal concentrations of PMA, convulxin, and thrombin is more or less identical. Also, all 3 stimulate PKD activation via PKC. This shows PKD as a common point of signal amplification downstream of tyrosine kinase- and Gq-coupled receptors. Convulxin-induced PKD activation was partially dependent on PI
3-kinase and entirely dependent on the Src kinases.48,64 Because activation of PLC PKD activation is transient in response to weak platelet agonists U46619 (present study) and ADP (data not shown). This is likely to be the result of these agonists' activating PKC only temporarily, as sustained PKC activity is required to maintain PKD activation. After treatment with convulxin, thrombin, and PMA, a subsequent addition of Ro 31-8220 led to a loss of PKD activity, suggesting that a phosphatase is active in platelets that can reverse PKD activation unless the PKC stimulus remains. Transient PKC activation was confirmed by measurements of phosphorylation of pleckstrin, an alternative PKC substrate in platelets. This transient regulation of PKD activation in response to weak agonists is unusual, as in other systems PKD activation is sustained.62,66 It is well established that synergy between signaling pathways can lead to platelet activation.55,58 Therefore, investigations were undertaken to identify whether synergy between signals from 2 separate receptors can activate PKD. The results presented here identify synergy through Gi/z and Gq and between Gi/z and GPVI leading to PLC and PKD activation in platelets. This synergy was also apparent when investigating PKD activation in response to ADP. Results show that activating either the P2Y1 receptor coupled to Gq or the P2Y12 receptor coupled to Gi/z was unable to activate PKD alone. This is different from the results observed for other Gq-coupled receptor agonists in this study (thrombin, U46619) and is likely to be due to the low number of P2Y1 receptors on the platelet surface. Previous studies have implied that PI 3-kinase is an important component of the synergy between Gi/z and Gq in the regulation of platelet aggregation,55,58,65 and this may also be the case for synergy at the level of PKC/PKD activation. These studies demonstrate activation of PKD downstream of GPVI and thrombin-dependent Gq signaling, in a PKC-dependent manner. PKD is a novel PKC substrate in platelets that phosphorylates a different recognition motif from that phosphorylated by PKC, enabling regulation of a different set of proteins from those targeted by PKC in platelets. A number of pathways that have been previously ascribed to PKC could in fact be regulated via PKD, because activation of PKC always leads to PKD activation. This could potentially include all of the roles involving PKC where the functional mechanisms remain ill-defined, such as aggregation and adhesion. Presently, however, there are no tools, such as selective inhibitors or mutant mice, to enable determination of the role of PKD function compared with the role of PKC in platelets. Future studies need to be performed to investigate whether PKC or PKD is responsible for a number of the effects downstream of PLC activation in platelets.
We are grateful to Dr Doreen Cantrell for pS916 and syntide-2, and for lots of helpful advice; to Ben Atkinson, who helped with the earlier studies; and to Dr Ulrica Marklund for lots of support and excellent advice.
Submitted August 27, 2002; accepted September 25, 2002.
Prepublished online as Blood First Edition Paper, October 10, 2002; DOI 10.1182/blood-2002-08-2384.
Supported by a British Heart Foundation (BHF) Studentship (M.J.S.) and a BHF Senior Research Fellowship (S.P.W.).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Margaret J. Stafford, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, United Kingdom; e-mail: margaret{at}bioch.ox.ac.uk.
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© 2003 by The American Society of Hematology.
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  M. Tan, F. Hao, X. Xu, G. M. Chisolm, and M.-Z. Cui Lysophosphatidylcholine Activates a Novel PKD2-Mediated Signaling Pathway That Controls Monocyte Migration Arterioscler Thromb Vasc Biol, September 1, 2009; 29(9): 1376 - 1382. [Abstract] [Full Text] [PDF]
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 M. Hoffmeister, P. Riha, O. Neumuller, O. Danielewski, J. Schultess, and A. P. Smolenski Cyclic Nucleotide-dependent Protein Kinases Inhibit Binding of 14-3-3 to the GTPase-activating Protein Rap1GAP2 in Platelets J. Biol. Chem., January 25, 2008; 283(4): 2297 - 2306. [Abstract] [Full Text] [PDF]
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E. Rozengurt, O. Rey, and R. T. Waldron Protein Kinase D Signaling J. Biol. Chem., April 8, 2005; 280(14): 13205 - 13208. [Full Text] [PDF]
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C. S. Buensuceso, A. Obergfell, A. Soriani, K. Eto, W. B. Kiosses, E. G. Arias-Salgado, T. Kawakami, and S. J. Shattil Regulation of Outside-in Signaling in Platelets by Integrin-associated Protein Kinase C{beta} J. Biol. Chem., January 7, 2005; 280(1): 644 - 653. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
in the regulation of
Bruton tyrosine kinase. In addition, PKC
associates with
phosphatidylinositol 3-kinase (PI 3-kinase)/
,
a family affair.
Mol Cell Endocrinol.
1989;65:1-11