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Published as doi: 10.1096/fj.07-102558.
 (The FASEB Journal. 2008;22:2768-2774.)
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The extreme C-terminal region of phospholipase Cβ1 determines subcellular localization and function; the "b" splice variant mediates {alpha}1-adrenergic receptor responses in cardiomyocytes

David R. Grubb, Oliver Vasilevski, Huy Huynh and Elizabeth A. Woodcock1

Cellular Biochemistry Laboratory, Baker Heart Research Institute, Melbourne, Victoria, Australia

1Correspondence: Baker Heart Research Institute, P.O. Box 6492, St. Kilda Road Central, Melbourne, 8008, Victoria, Australia. E-mail: liz.woodcock{at}baker.edu.au


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Phospholipase Cβ1 (PLCβ1) exists as two splice variants, PLCβ1a (150 kDa) and PLCβ1b (140 kDa), which differ only in their C-terminal sequences of 64 and 31 amino acids, respectively. The 3 C-terminal amino acid residues of PLCβ1a comprise a PDZ-interacting domain, whereas the PLCβ1b sequence has no PDZ-interacting domain but contains unique proline-rich domain 5 residues from the C terminus. PLCβ1a is localized in the cytoplasm, whereas PLCβ1b targets to the sarcolemma and is enriched in caveolae. Deletion of 3 amino acids from the C terminus of PLCβ1b did not alter its sarcolemmal localization, but deletion of the entire unique 31 amino acid sequence caused cytosolic localization. A myristoylated 10 amino acid peptide from the C terminus of PLCβ1b selectively dissociated N-terminally GFP-tagged PLCβ1b from the sarcolemma and inhibited PLC responses to {alpha}1-adrenergic agonists, with a half maximal effective concentration of 12 ± 1.6 µM (mean±SE, n=3). A similar peptide from PLCβ1a was without effect at concentrations below 100 µM. Thus, the extreme C-terminal sequences of the PLCβ1 splice variants determine localization and, thus, function. In cardiomyocytes, responses initiated by {alpha}1-adrenergic receptor activation involve only PLCβ1b, and the selective targeting of this splice variant to the sarcolemma provides a potential therapeutic target to reduce hypertrophy, apoptosis, and arrhythmias.—Grubb, D. R., Vasilevski, O., Huynh, H., and Woodcock, E. A. The extreme C-terminal region of phospholipase Cβ1 determines subcellular localization and function; the "b" splice variant mediates {alpha}1-adrenergic receptor responses in cardiomyocytes.


Key Words: proline-rich domain • Gq • PLC • myristoylated peptides • signaling specificity


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
THE HETEROTRIMERIC G PROTEIN (Gq) is an initiator of a number of responses in cardiomyocytes, important among which are pathological hypertrophic growth as well as apoptosis at very high levels of activation (1) . Whereas the details of the signaling pathways involved in these responses remain unclear, it is likely that these involve activation of phospholipase Cβ (PLCβ) isoforms by the {alpha} subunits of Gq, following activation of appropriate receptors (2) . Activation of PLC increases the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) to generate inositol 1,4,5-trisphosphate (Ins[1,4,5]P3) and sn-1,2-diacylglycerol (DAG), which have the capacity to regulate intracellular Ca2+ and protein kinase C activity as well as Ca2+ entry via transient receptor potential (Trp) channels. There are 4 subtypes of PLCβ, classified as PLCβ1–4, of which PLCβ1, β2, and β4 are expressed as two splice variants (3 4 5) . The PLCβ1 subtype has been shown to respond primarily to activated G{alpha}q, whereas PLCβ2 responds to β{gamma} subunits released following activation of Gi-coupled receptors and, to a lesser extent, to G{alpha}q. PLCβ3 responds effectively both to G{alpha}q and to Gβ{gamma} (6) . PLCβ4, which has been studied less than the other family members, is also activated by G{alpha}q subunits. Only PLCβ1 and PLCβ3 are expressed in rat cardiomyocytes (7) .

The two splice variants of PLCβ1, PLCβ1a and PLCβ1b, differ only in the extreme C-terminal regions and have molecular masses of 150 and 140 kDa, respectively (8 , 9) . The splice-specific sequences comprise 64 of 1216 amino acids for PLCβ1a and 31 of 1173 amino acids for PLCβ1b, thus representing a minor part of the sequence. The extreme C-terminal 3 amino acids of PLCβ1a form a PDZ-interacting domain that is potentially involved in localization and thus may be important for the functioning of the enzyme in some cell types (10) . The C-terminal 10 amino acids of PLCβ1b, in contrast, do not include a PDZ-interacting domain but instead contain a proline-rich sequence that can potentially target to SH3 domains or WW domains (Fig. 1 ). Thus, the two splice variants would be predicted to interact with different proteins, possibly have different cellular localizations, and may be functionally diverse. The common sequence of PLCβ1 has a nuclear localization motif, and both splice variants have been shown to accumulate in the nucleus under some conditions (9 , 11) . PLCβ1b is particularly enriched in nuclear fractions from erythorleukemia cells (12) . Phosphorylation of PLCβ1b on Ser-982 by ERK1/2 following activation of cell surface growth factor receptors (13) has been associated with enhanced activity and nuclear localization of PLCβ1b, where it has been suggested to play a role in cell cycle control, although it remains unclear exactly how this is mediated (11 , 13 , 14) . Nuclear-localized PLC activity appears to be most often observed in undifferentiated cells (11) and, on that basis, is unlikely to apply to cardiomyocytes, even when derived from neonatal animals.


Figure 1
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  		Figure 1. The two splice variants of PLCβ1. PH domain (PH); EF hand (EF); the X-box (X) and Y-box (Y) regions comprising the catalytic domain; a C2 domain (C2); the site of interaction with G{alpha}q (G{alpha}q); the putative nuclear localization sequence (NLS) (1056–1070; ref. 12 ); and the different C-terminal 10-amino acid sequences of the two splice variants, containing the PDZ-interacting domain of PLCβ1a and the proline-rich domain of PLCβ1b.

Neonatal rat ventricular myocytes (NRVMs) express both splice variants of PLCβ1 (3 , 7) as well as PLCβ3. We have previously shown that responses to {alpha}1-adrenergic receptor activation exclusively involve PLCβ1, even though PLCβ3 is also expressed (3) , and both subtypes respond similarly to activated G{alpha}q. In subsequent experiments, we found that PLCβ1, but not PLCβ3, was enriched in caveolae, implying differing roles for these two isoforms in cardiomyocytes (15) . In the current study, we examined the roles of the two splice variants of PLCβ1 in cardiomyocytes. We found that PLCβ1b in cardiomyocytes is selectively localized to the sarcolemma, is mostly nuclear excluded, and appears to be the major mediator of {alpha}1-adrenergic receptor Gq-initiated responses.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Preparation of NRVMs
Ventricular myocytes were prepared from 1- to 2-day-old Sprague-Dawley rats using repeated pancreatin/collagenase digestion, followed by separation from nonmyocytes using discontinuous Percoll gradients, as described previously (16) . Cells were plated at 400/mm2 on gelatin-coated plates in medium comprising Dulbecco modified Eagle medium (DMEM)/M-199 (4:1) supplemented with 10% horse serum, 5% fetal calf serum, 50 U/mL penicillin, and 50 µg/mL streptomycin sulfate. After plating, cells were maintained in defined medium comprising DMEM, insulin (50 µg/mL), transferrin (10 µg/mL), sodium selenate (30 nM), bromodeoxyuridine (BrdU; 0.1 mM), and antibiotics. Cells were used within 5 days of preparation. BrdU was omitted after 3 days.

Constructs and adenoviruses
Adenoviruses expressing G{alpha}q, G{alpha}q(Q209L), and G{alpha}q(Q209L, D243A, N244A, E245A) (Gq[QL, DNE]) were provided by Dr. Joan Heller Brown (Department of Pharmacology, University of California, San Diego). Expression plasmids encoding enhanced green fluorescent protein (EGFP) -PLCβ1a and EGFP-PLCβ1b (N-terminal EGFP) were provided by Dr. P. G. Suh (Department of Life Science, Pohang University of Science and Technology, Kyungbuk, South Korea). Cardiomyocytes were transfected using Lipofectamin 2000 (Invitrogen, Melbourne, VIC, Australia) according to the manufacturer’s instructions.

Confocal microscopy
Confocal images of NRVMs were captured using a Zeiss Meta510 LSM (Carl Zeiss, Oberkochen, Germany) after transfection with either pEGFP-PLCβ1a, pEGFP-PLCβ1b, pEGFP-PLCβ3, or pEGFP (vector control). A minimum of 20 transfected cells per plate were imaged from 6 independent experiments carried out in duplicate. For experiments involving the addition of myristoylated peptides to live cells, a single myocyte was followed for 15 min, with images captured every minute. Images were collected as 0.7 µm optical sections.

Immunoprecipitation
NRVMs were treated with 1 mM dithiobis(succinimidyl propionate) for 1 min, followed by chilling on ice. The cells were harvested in ice-cold lysis buffer containing Hepes (50 mM, pH 7.4), NaCl (130 mM), MgCl2 (1 mM), KH2PO4 (40 mM), Triton X-100 (1% v/v), Nonidet P-40 (5% v/v), glycerol (15% v/v), and BSA (0.2% w/v), plus protease inhibitor cocktail mix containing PMSF, aprotinin, leupeptin A, and pepstatin A. Samples were precleared with protein A sepharose for 1 h prior to the addition of 2 µg anti-G{alpha}q antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and 40 mg protein A sepharose to 100–200 µg of protein from the precleared lysate. Immunoprecipitated material was harvested by centrifugation and washed sequentially as described previously (17) .

Western blotting
Proteins were separated on gradient SDS-PAGE (7.5–15% acrylamide) (18) , electrophoretically transferred to nitrocellulose membranes (Schleicher and Schuell, Dassel, Germany), and stained with Ponceau-S (Sigma-Aldrich, Sydney, NSW, Australia). Membranes were blocked using 5% skim milk in PBS plus 0.05% v/v Tween-20 prior to incubation with either anti-PLCβ1 (1 µg/mL; Santa Cruz) or anti-G{alpha}q antibodies (1/250; Santa Cruz). HRP-conjugated secondary antibodies and ECL plus (Amersham Life Sciences, Castle Hill, NSW, Australia) were used to detect proteins of interest.

Measurement of PLC activity in NRVMs
NRVMs were labeled for 2 days with [3H]inositol (5 µCi/mL) in inositol-free-DMEM supplemented with insulin (50 µg/mL), transferrin (10 µg/mL), sodium selenate (30 nM), BrdU (0.1 mM), and antibiotics; washed with unlabeled medium; and pretreated for 10 min with 10 mM LiCl and 1 µM propranolol in DMEM prior to the addition of agonists. [3H]inositol phosphates were extracted with ice-cold 5% (w/v) trichloroacetic acid (TCA), EDTA (2.5 mM), and phytic acid (5 mM), and the supernatants subsequently treated with a 1:1 mixture of 1,1,2-trichlorotrifluoroethane:tri-n-octylamine to remove remaining TCA. The aqueous phase containing [3H]inositol phosphates was subjected to chromatography on Dowex 1 columns for total [3H]inositol phosphate measurement (3) .

Preparation of the caveolar fractions
Caveolar fractions were prepared by immunoprecipitation of light lipid raft fractions with anti-caveolin antibody, as described previously (15) .

Materials
Myristoylated C-terminal peptides Myr-NPGKEFDTPL-NH2 for PLCβ1a and Myr-TPPNPQALKW-NH2 for PLCβ1b were synthesized by Auspep (Melbourne, VIC, Australia). [3H]Inositol was purchased from Auspep. Antibodies to PLCβ1, PLCβ3, and G{alpha}q were from Santa Cruz. Anti-caveolin antibody (pan) was from Transduction Laboratories (Lexington, KY, USA). Cell culture media were from Life Technologies, Inc. (Melbourne, VIC, Australia).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
PLCβ1b, but not PLCβ1a, localizes to the sarcolemma in NRVMs
NRVMs express PLCβ3 as well as two splice variants of PLCβ1, PLCβ1a and PLCβ1b, of 150 and 140 kDa, respectively, that differ only in their extreme C-terminal sequences (64 and 31 amino acids, respectively). The two splice variants of PLCβ1 are expressed at similar levels in cellular extracts prepared from NRVMs, as evaluated by Western blots (3) . In some other cell types, PLCβ1a is expressed in the cytosol and plasma membrane, whereas the "b" splice variant is concentrated in the nucleus, where it appears to be involved in cell cycle control (12) . Because of the very small sequence differences between them and the fact that the C-terminal proline-rich sequence of PLCβ1b is unsuitable for antibody generation, endogenous PLCβ1a and PLCβ1b can only be distinguished by their differing molecular masses. Therefore, to define the localizations of the endogenous proteins, it was necessary to prepare cell fractions and identify PLCβ1a and PLCβ1b by SDS-PAGE and Western blotting. Membrane and cytosolic fractions were prepared, as described, from harvested NRVMs, and the PLCβ subtypes and splice variants were measured. PLCβ1b was found in both the membrane and cytosolic fractions, whereas PLCβ1a was only detectable in the cytosolic fraction (Fig. 2 A). PLCβ3 was also detected only in the cytosolic fraction (Fig. 2A ). To further define the membrane fraction containing PLCβ1b, light lipid raft fractions prepared using buoyant sucrose density gradients were immunoprecipitated using anti-pan-caveolin antibodies to enrich in caveolae, as described previously (15) . Western blot analysis identified PLCβ1b, but not PLCβ1a, as being highly enriched in caveolar fractions (Fig. 2B ) and containing no detectable PLCβ3 (11) . Stimulation with the {alpha}1-adrenergic receptor agonist phenylephrine (50 µM, plus 1 µM propranolol) for either 2 or 20 min did not alter the subcellular localization of any of the PLCβ subtypes, as reported previously (11) .


Figure 2
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  		Figure 2. PLCβ1b is enriched in the sarcolemma in NRVMs. A) Membrane and cytosolic fractions were prepared from NRVMs, and the content of endogenous PLCβ1 or PLCβ3 was assessed by Western blot (50 µg protein loaded). The experiment was performed 3 times with similar data. Arrows indicate molecular mass (kDa). B) PLCβ1b is enriched in the caveolar fractions. Caveolar fractions were prepared as described, and PLCβ1 content was measured in the caveolar and nonraft fractions (non-LLR); 10 µg protein was loaded in the caveolar and nonraft lanes and 50 µg protein in the total lysate lanes. Values are mean ± SE density/µg protein; n = 3. ***P < 0.001 relative to nonraft fractions. C) NRVMs expressing N-terminal EGFP-tagged PLCβ1a, PLCβ1b, PLCβ3, or EGFP alone were examined by confocal microscopy. Subcellular localization of EGFP-tagged PLCβ subtypes shown here are typical of >95% of the transfected NRVM population. PLCβ1a, 97 cells counted, 97% cytosolic; PLCβ1b, 94 cells counted, 95% sarcolemmal.

In the next set of experiments, N-terminal fusion proteins EGFP-PLCβ1a, EGFP-PLCβ1b, and EGFP-PLCβ3 were expressed in NRVMs, and the subcellular localization was examined by confocal microscopy. As shown in Fig. 2C , EGFP-PLCβ1a and EGFP-PLCβ3 were distributed throughout the cytosol and were largely excluded from the nucleus. EGFP-PLCβ1b, in contrast, was concentrated at the sarcolemma, although it also was present in the cytosol, but to a lesser extent than for EGFP-PLCβ1a or EGFP-PLCβ3. Neither EGFP-PLCβ1a nor EGFP-PLCβ3 showed any significant association with the sarcolemma. Like EGFP-PLCβ1a and EGFP-PLCβ3, EGFP-PLCβ1b was largely nuclear excluded, despite the fact that it has a nuclear localization sequence and has been reported to localize to nuclei in other cell types (9) . Addition of the {alpha}1-adrenergic agonist phenylephrine did not alter the distribution of any of the PLCβ constructs.

The unique C-terminal sequence of PLCβ1b is responsible for its sarcolemmal localization
The different localizations of PLCβ1a and PLCβ1b implied that the unique C-terminal tails were responsible for localization of the two splice variants. To examine this directly, the entire C-terminal tail of 31 amino acids was removed from the EGFP-PLCβ1b fusion protein. Deletion of the PLCβ1b-specific C-terminal tail resulted in the loss of sarcolemmal association (Fig. 3 ) and resulted in a subcellular localization pattern identical to that of EGFP-PLCβ1a. In contrast, deletion of either the C-terminal PDZ-interacting domain from PLCβ1a (PLCβ1a {Delta}PDZ) or the 3 C-terminal amino acids from EGFP-PLCβ1b (PLCβ1b {Delta}3) had no obvious effect on localization when compared with EGFP-PLCβ1a and EGFP-PLCβ1b, respectively.


Figure 3
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  		Figure 3. The unique C-terminal tail of PLCβ1b determines sarcolemmal localization. NRVMs expressing N-terminal EGFP-tagged PLCβ1a and PLCβ1b C-terminal deletion mutants were examined by confocal microscopy. Top panels: PLCβ1a and PLCβ1a with deletion of the 3 C-terminal residues. Middle panels: PLCβ1b and PLCβ1b with 3 C-terminal resides deleted. Bottom panels: PLCβ1 with the entire unique C-terminal sequences deleted.

G{alpha}q associates preferentially with PLCβ1b in NRVMs
It has recently been reported that G{alpha}q is associated with PLCβ1, even in unstimulated cells (19) . We next examined whether both PLCβ1 splice variants coupled equally to Gq in NRVMs. NRVMs were treated with adenoviruses expressing wild-type G{alpha}q for 2 days; lysates were prepared and immunoprecipitated with anti-G{alpha}q antibody, and bound proteins were separated by SDS-PAGE. Western blot analysis identified that PLCβ1b, but not PLCβ1a, immunoprecipitated with G{alpha}q in unstimulated NRVMs (Fig. 4 bottom panel). In agreement with previous studies (19) , addition of an {alpha}1-adrenergic agonist did not alter the association between G{alpha}q and PLCβ1b.


Figure 4
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  		Figure 4. PLCβ1b immunoprecipitates with G{alpha}q. NRVMs were infected with adenoviruses expressing G{alpha}q-WT, G{alpha}q(Q209L) (constitutively active); or G{alpha}q(Q209L, D243A, N244A, E245A), a constitutively active mutant with reduced ability to activate PLCβ. Top panel: PLC activity in [3H]inositol-labeled, adenovirus-treated NRVMs, measured as [3H]inositol phosphate accumulation over 20 min, mean ± SE counts per minute (CPM); n = 3. ***P < 0.001 relative to LacZ. {dagger}{dagger}{dagger}P < 0.001 relative to Gq(Q209L). Bottom panel: lysates were prepared and immunoprecipitated with anti-G{alpha}q antibody, separated on SDS-PAGE, and blotted with anti-PLCβ1 antibody. The experiment was performed 4 times with similar data.

The functional significance of the association of G{alpha}q with PLCβ1b was further examined by assessing the interaction of PLCβ1b with G{alpha}q mutants that have altered ability to activate PLCβ. NRVMs were treated with adenoviruses expressing constitutively active G{alpha}q (G{alpha}q[Q209L]) or G{alpha}q(Q209L) with 3 alanine substitutions that reduce PLCβ association G{alpha}q(Q209L, D243A, N244A, E245A) (QL-DNE) (2) . As shown in Fig. 4 , wild-type G{alpha}q (G{alpha}qWT) and constitutively active G{alpha}q(Q209L) both immunoprecipitated with PLCβ1b, but not PLCβ1a, and induced a significant increase in PLC activity. In contrast, G{alpha}q(QL-DNE) did not immunoprecipitate with PLCβ1b.

PLCβ1b C terminal peptide inhibits PLC responses to {alpha}1-adrenergic receptor activation
Initially, we attempted to reduce expression of both PLCβ1 splice variants using small interfering RNA sequences from the common translated region. Overall, 25 different sequences were attempted, including published sequences (20) . However, none of these effectively reduced PLCβ1 protein expression, although some sequences reduced mRNA as assessed by quantitative real-time polymerase chain reaction (results not shown), likely partly because of the limited time in which NRVMs remain viable in culture.

As an alternative approach, myristoylated 10-amino acid peptides identical to the C-terminal sequences of PLCβ1a and PLCβ1b were used. NRVMs were labeled with [3H]inositol prior to the addition of either the PLCβ1a (Myr-NPGKGFNTPL-NH2) or PLCβ1b (Myr-TPPNPQALKW-NH2) C-terminal peptide. PLC responses were then determined as total [3H]inositol phosphate accumulation over 20 min after addition of the {alpha}1-adrenergic agonist norepinephrine (100 µM), in the presence of 1 µM propranolol and 10 mM LiCl. As shown in Fig. 5 (left panel), the PLCβ1b peptide effectively inhibited PLC activity in response to {alpha}1-adrenergic receptor activation in a dose-dependent manner, with an EC50 of 12 ± 1.6 µM (n=3). In contrast, the PLCβ1a peptide had no significant effect on PLC activity.


Figure 5
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  		Figure 5. The C-terminal peptide from PLCβ1b inhibits {alpha}1-adrenergic receptor-activated PLC. Left panel: NRVMs were labeled with [3H]inositol and subsequently treated with a myristoylated 10-amino acid peptide (0–200 µM) corresponding to the C-terminal sequences of either PLCβ1a or PLCβ1b. Norepinephrine (NE; 100 µmol/L, 1 µM propranolol, and 10 mM LiCl) was added for 20 min, and total [3H]inositol phosphates were extracted and quantified. Values are mean ± SE fold PLC activation by NE; n = 3. PLCβ1a peptide, blue circles; PLCβ1b peptide, red squares. Values were significant at 5 µM PLCβ1b peptide (P<0.05) and 1 µM (P<0.01) at higher concentrations. Right panel: N-terminal EGFP-fusion proteins of PLCβ1a and PLCβ1b were expressed in NRVMs. Myristoylated C-terminal peptides from PLCβ1a and PLCβ1b (200 µM) were added, and the localization of EGFP-PLCβ1a and EGFP-PLCβ1b was observed over 5 min by confocal microscopy. EGFP controls are shown. The experiment was performed 12 times with similar results.

The effect of the C-terminal peptides on the localization of the PLCβ1 splice variants was assessed using EGFP fused to the N-terminal of PLCβ1a and PLCβ1b expressed in NRVMs. At 24 h post-transfection, NRVMs were treated with 200 µM of either the PLCβ1a or PLCβ1b C-terminal peptide, and the localization of EGFP-PLCβ1a or EGFP-PLCβ1b was examined in individual cells by confocal microscopy for 5 min. As shown in Fig. 5 (right panels), the PLCβ1b C-terminal peptide induced the dissociation of EGFP-PLCβ1b from the sarcolemma into the cytosol. The PLCβ1b C-terminal peptide did not alter the localization of EGFP-PLCβ1a. There was also no effect on the distribution of EGFP or EGFP-PLCβ3 (data not shown). In contrast, the C-terminal PLCβ1a peptide did not alter the localization of EGFP, EGFP-PLCβ1a, EGFP-PLCβ1b (Fig. 5 , right panels), or EGFP-PLCβ3 (not shown).

PLCβ1a expression is lost during postnatal development
These data suggest that PLCβ1b, and not PLCβ1a, is responsible for mediating responses to Gq-coupled receptors in NRVMs. Unlike cardiomyocytes derived from adult animals, NRVMs are not fully differentiated. When adult rat cardiomyocytes were compared with the neonatal cells, cardiomyocytes from adult rats expressed only the PLCβ1b splice variant (Fig. 6 ) and not PLCβ1a. PLCβ3 expression remained unchanged between neonatal and adult cardiomyocytes. Thus, at least in rats, fully differentiated cardiomyocytes express only the b splice variant of PLCβ1. This result supports the conclusion that PLCβ1b is the functionally active PLCβ1 splice variant in cardiomyocytes and implies the regulated splicing of PLCβ1 during the maturation of the cardiomyocyte.


Figure 6
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  		Figure 6. Adult rat cardiomyocytes express only the b splice variant of PLCβ1. Adult rat ventricular myocytes (ARVMs) were prepared from 4 different rats; extracts were prepared, and the content of the splice variants of PLCβ1 as well as PLCβ3 were examined by SDS-PAGE and Western blotting. Arrows indicate splice variants of PLCβ1. The profile from neonatal rats (NRVM) is shown for comparison.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In hearts and cardiomyocytes, increased activity of the heterotrimeric Gq initiates pathological hypertrophic growth responses and, with protracted activation, causes apoptosis (21) . Similarly, inhibiting Gq activity prevents the development of pressure overload hypertrophy in vivo and delays the development of heart failure (22 , 23) . Downstream signaling pathways initiated by Gq are complex, and the intermediates responsible for these critical actions of Gq have not been unequivocally identified. Activated G{alpha}q binds directly to its immediate effector, one of the PLCβ subtypes. This action stimulates PIP2 hydrolysis and the generation of the Ca2+-mobilizing messenger Ins(1,4,5)P3, as well as DAG, that can activate conventional PKC isoforms (6) and several of the transient receptor potential (Trp) channels, TrpC3, Trp6C, and TrpC7 (24) . TrpC4 and TrpC5 also appear to be regulated by PLC activity, but the mechanism is not known (25) . Recent evidence points to a critical role for TrpC3 and TrpC6 in the Ca2+ responses that underpin pathological heart growth (26 , 27) . Despite this, a requirement for PLC activation in Gq-initiated hypertrophy has not been unequivocally demonstrated, and the subtype of the PLC involved has not been established. This study was undertaken to identify the PLC enzyme responsible for mediating responses to {alpha}1-adrenergic receptor-Gq activation in the heart, with a view to selectively inhibiting Gq-PLC signaling in the heart (24) .

PLC enzymes exist as a number of different gene products, PLCβ, {gamma}, {delta}, {epsilon}, and {zeta}, that are activated by different mechanisms (6 , 28 , 29) . In addition, a number of these PLCs exist as more than one splice variant. Responses to activated G{alpha}q and, to a lesser extent, Gβ{gamma} are mediated primarily by one or more of the four PLCβ subtypes, (PLCβ1–4) (30) . All of the PLCβ enzymes are high-molecular-mass proteins (130–150 kDa), and all have multiple potential localization and regulatory domains. The PLCβ1 and PLCβ3 enzymes, which are expressed in cardiomyocytes (7) , contain a pleckstrin homology (PH) domain, C2 domains, and a G{alpha}q-binding domain, as well as catalytic X-box and Y-box domains (Fig. 1) . Gβ{gamma} and the monomeric Gqs Rac and Rho can interact at or close to the PH domain region (31 32 33) .

Unlike PLC{delta} subtypes, the PH domains of the PLCβ enzymes are not sufficiently strong to facilitate membrane localization by binding PIP2 (32) . This raises questions as to how the membrane association required for receptor regulation is achieved. One possible explanation is that activation is achieved by binding activated G{alpha}q following receptor ligation. Binding to G{alpha}q might serve to localize the PLC to the sarcolemma, because G{alpha}q is concentrated in this fraction. However, the two splice variants of PLCβ1 have identical G{alpha}q-interacting domains and thus would be expected to bind G{alpha}q equally. Here we report that only PLCβ1b was localized to the sarcolemma in NRVMs (Fig. 2) . Thus, another mechanism is required to facilitate sarcolemmal association of the PLC. As the only sequence difference between the a and b splice variants of PLCβ1 are in the extreme C-terminal, the signal for localization must reside in this region. In agreement with this, deletion of the unique C-terminal tail resulted in a loss of sarcolemmal localization (Fig. 3) . PLCβ3 and the splice variants PLCβ1a and PLCβ2a possess a 3-amino acid C-terminal PDZ-interacting domain, which can result in the membrane localization of PLCβ variants if the appropriate PDZ domain proteins are expressed (34) . In contrast, PLCβ1b has no C-terminal PDZ-interacting domain, and deleting the 3 C-terminal amino acids did not alter its localization (Fig. 3) . Instead, PLCβ1b contains a proline-rich sequence in the C-terminal sequence (Fig. 1) . Rather than binding PDZ-interacting domain proteins, proline-rich sequences bind SH3 domains and WW domains (35 , 36) . To our knowledge, this is the first demonstration of specific localization of PLCβ1b via its unique C-terminal domain.

As implied by its specific sarcolemmal localization, PLCβ1b appeared to be the splice variant mediating responses to activated Gq. Immunoprecipitation studies showed that Gq was bound to PLCβ1b but not to PLCβ1a (Fig. 4) . Interestingly, G{alpha}q was bound to PLCβ1b even in the absence of a stimulus, and stimulating Gq-coupled receptors through the addition of the {alpha}1-adrenergic receptor agonist phenylephrine did not increase the association. A similar association of PLCβ1 with G{alpha}q in unstimulated cells has been reported previously, but the splice variant involved was not identified. The region of PLCβ1 that interacts with G{alpha}q is not in the extreme C-terminal sequence (6 , 37 , 38) , so G{alpha}q would be expected to interact with the two splice variants similarly. Thus, it is more likely that PLCβ1b is targeted to the sarcolemma by its unique C-terminal domain and that this localization facilitates the association with G{alpha}q.

C-terminal peptides from the two PLCβ1 splice variants, rendered cell permeable by myristoylation, were used to reduce sarcolemmal association of the PLC. The C-terminal peptide from the sequence of PLCβ1b proved to be a very powerful inhibitor of {alpha}1-adrenergic receptor-mediated PLC responses, whereas the peptide derived from the C terminus of PLCβ1a caused no inhibition. The peptides used in these studies were only 10 amino acids in length, and it seems likely that the selective targeting by the PLCβ1b peptide involves the C-terminal proline-rich domain. This probability would imply that a sarcolemmal protein specifically binds the unique C-terminal region of PLCβ1b to target PLCβ1b to the sarcolemma and to the caveolae, where it associates with G{alpha}q. It would be expected that PLCβ1b would interact with as yet unknown SH3 domain or possibly WW domain-containing proteins rather than PDZ domain proteins. On this basis, it may be possible to target PLC activation in cardiomyocytes by selectively interfering with this interaction.


   ACKNOWLEDGMENTS
 
This work was supported by grants from the National Health and Medical Research Council of Australia (268921, 317801, and 317802), a research fellowship to E.A.W. (317803), and an Australian postgraduate award to O.V. We thank Dr. J. H. Brown (Pharmacology, University of California, San Diego) for the Gq-expressing adenoviruses and Dr. P. G. Suh (Department of Life Sciences, Pohang University of Science and Technology, Kyungbuk, South Korea) for the plasmids expressing PLCβ1 splice variants. Dr. R. Andrews (Pathology and Immunology Department, Monash University, Clayton, VIC, Australia) provided invaluable assistance with protein-protein interaction domains.

Received for publication January 21, 2008. Accepted for publication March 6, 2008.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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