Activation of Phospholipase C-β1 via Gαq/11during Calcium Mobilization by Calcitonin Gene-related Peptide*

Interaction of calcitonin gene-related peptide (CGRP) with its receptors leads to stimulation of adenylyl cyclase and/or phospholipase C (PLC). While regulation of adenylyl cyclase is thought to involve the G-protein Gs, it is not known whether activation of PLC results from coupling the receptor to Gq family proteins or whether βγ subunits released from receptor-activated Gs activate PLC. We used human bone cells OHS-4 bearing CGRP receptors in which CGRP activates only the PLC signaling pathway to determine how CGRP acts. CGRP increased the concentration of intracellular calcium ([Ca2+] i ) within 5 s via a Ca2+ influx through voltage-gated calcium channels and by mobilizing calcium from the endoplasmic reticulum. The activation of effectors, like PLC coupled to G-proteins, is the early event in the pathway leading to inositol 1,4,5-trisphosphate formation, which is responsible for Ca2+ mobilization. Western blotting demonstrated a range of PLC-β isoforms (β1, β3, β4, but not β2) and G-proteins (Gαq/11 and Gαs). Only phospholipase C-β1 is involved in the mobilization of Ca2+ from the endoplasmic reticulum of Fura-2-loaded confluent OHS-4 cells and the formation of inositol 1,4,5-trisphosphate by CGRP; PLC-γ have no effect. Activation of PLC-β1 by CGRP involves the Gαq/11 subunit, which is insensitive to pertussis toxin, but not Gβγ subunits. We therefore believe that CGRP causes the activation of two separate G-proteins.

tic OHS-4 cells, which possess CGRP receptors (19), CGRP does not stimulate adenylyl cyclase as do other cell types (20,21), but it does increase intracellular calcium. This suggests that the CGRP receptor activates different G-proteins than do other calciotropic peptide hormones (22).
There are many similar heterotrimeric G-proteins that transduce a signal from a hormone-bound receptor to a variety of downstream effector molecules (23)(24)(25). Mammals have several types of G-proteins, which form the ␣ subunit and the ␤ and ␥ subunits. These include the G q , G s , G i /G o , and G 12 subfamilies, which are classified on the basis of amino acid sequence of the ␣ subunits. G-proteins are also divided traditionally into two types based on their sensitivity to Bordetella pertussis toxin (PTX) (26). The PTX-sensitive G-proteins are inactivated by ADP-ribosylation of the ␣ subunit. This group includes members of the G i and G o subfamily. G s -and G iproteins were originally purified as mediators of adenylyl cyclase pathway (27). PTX-insensitive G-proteins are resistant to ADP-ribosylation and include members of the G q subfamily (27). The ␣ subunits of these proteins activate phospholipase C-␤ isoenzymes (28), but not phospholipases (PLCs) C␥ or C␦ (29 -33). PLC-␤ isoenzymes may be stimulated by both G-protein ␣ subunits of the G q family and by free ␤␥ subunits (34 -36).
PLC can thus be stimulated via receptors with the dual signaling properties described above. Binding of the ligand to the receptor either stimulates adenylyl cyclase via G␣ s , and phospholipase C is activated by ␤␥ subunits released from activated G s , or the receptor couples to multiple G proteins leading to the activation of adenylyl cyclase and PLC via G s and ␣ subunits of the G q family (37). The latter route appears to apply to the human TSH receptor (38), and the turkey erythrocyte ␤-adrenergic receptor (39). The present study identifies and characterizes the PLC isoform involved in the mobilization of Ca 2ϩ from the endoplamic reticulum and inositol 1,4,5 trisphosphate formation of bone OHS-4 cells in response to CGRP, and its regulation by G-protein ␣ and ␤␥ subunits.
Human Cell Culture-The human bone cell line OHS-4, which exhibits most of the osteoblast phenotype (40), was kindly provided by Drs. B. Fournier and P. Price. Cells were grown on rectangular glass coverslips or in Petri dishes (100 cm 2 ) for 4 days in Dulbecco's modified * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Eagle's essential medium supplemented with 10% heat-inactivated fetal calf serum. Cells were then incubated for 72 h in Dulbecco's modified Eagle's essential medium containing 1% heat-inactivated fetal calf serum and transferred to serum-free medium 24 h before use.
Calcium The values for R max and R min were calculated from measurements using 25 M digitonin and 4 mM EGTA and enough Tris base to raise the pH to 8.3 or higher. Each measurement on Fura-2-loaded cells was followed by a parallel experiment under the same conditions with cells not loaded with Fura-2.
We first studied the direct effects of CGRP (0.1-100 nM) on [Ca 2ϩ ] i in OHS-4 cells. We then investigated whether the effects of CGRP on [Ca 2ϩ ] i were due to an influx of Ca 2ϩ from the extracellular milieu and/or Ca 2ϩ mobilization from intracellular stores. Two types of block-  Cell homogenates were prepared as described, and 35-g aliquots were separated by electrophoresis on SDS-polyacrylamide gel, transferred to nitrocellulose membranes, and probed with antibodies against the various PLC-␤ isoforms (arrows). The concentrations of anti-PLC antibodies were 0.1 g/ml for PLC-␤1, 0.5 g/ml for PLC-␤2, 1 g/ml for PLC-␤3, and 0.5 g/ml for PLC-␤4. Lines indicate the molecular masses of the standards (Std) from top to bottom: myosin, 199 kDa; ␤-galactosidase, 120 kDa; bovine serum albumin, 87 kDa; and ovalbumin, 48 kDa.

FIG. 5. G-proteins in confluent OHS-4 cells.
Aliquots (20, 30, and 50 g) of each homogenate were separated by electrophoresis on SDSpolyacrylamide gel, transferred to nitrocellulose membranes, and probed with antibodies against various ␣ subunits of G-proteins (arrows). The concentrations of anti-G␣ q/11 and anti-G␣ s antibodies were 0.2 g/ml and 0.5 g/ml, respectively. Lines indicate the molecular masses of the standards from top to bottom: phosphorylase B, 105 kDa; bovine serum albumin, 82 kDa; ovalbumin, 49 kDa; carbonic anhydrase, 33.3 kDa; and soybean trypsin inhibitor, 28.6 kDa.
ing experiments were performed. One involved adding a small excess of EGTA (2 mM), a chelator of extracellular calcium, to the medium in the cuvette and incubating for 30 s before adding CGRP. The second involved blocking of L-type Ca 2ϩ channels with 1 M nifedipine and verapamil. We investigated how much of the transient increase in [Ca 2ϩ ] i was due to Ca 2ϩ release from the endoplasmic reticulum using neomycin, which inhibits PLC by binding to phosphoinositides (42), and U-73122, which directly inhibits the PLC involved in the hydrolysis of phosphatidylinositol 4,5-bisphosphate (43). We then determined whether different PLC-␤ isoenzymes are involved in the effects of CGRP by incubating OHS-4 cells for 5 min with 20 g/ml saponin plus excess anti-PLC antibody, anti-G-protein antibody, or nonimmune rabbit serum (44). Cells were washed twice to remove saponin and incubated with the anti-PLC antibody, anti-G-protein antibody, or nonimmune rabbit serum for 1 h at 37°C. 1 M Fura-2/AM was added for the last 20 min of incubation. In some experiments, anti-PLC-␤1 antibody and anti-G␣ q/11 antibody were set up in competition with the antigen against which they were produced, or with the antigens corresponding to the other anti-PLC antibodies for 2 h at room temperature (antibody: peptide 1:10 or 1:100, according to the specifications of the manufacturer), prior to use. CGRP was used at the concentration that gave the greatest increase in [Ca 2ϩ ] i in confluent OHS-4 cells. The G-protein involved in the activity of CGRP was identified by incubating the cells with 100 ng/ml PTX for 16 h. Fura-2/AM loading and [Ca 2ϩ ] i measurements were carried out with the toxin.
Cell Homogenates and Membranes-Cells were washed three times with ice-cold phosphate-buffered saline, pH 7.4, and scraped off into ice-cold extraction buffer (20 mM Tris-HCl, pH 7.5, 0.5 mM EGTA, 2 mM EDTA, 0.6 mM pepstatin, 0.5 mM benzamidine, 0.1 mM leupeptin, 2 mM phenylmethylsulfonyl fluoride, 0.125 mM aprotinin, and 1 mM dithiothreitol). They were sonicated on ice twice for 20 s each at 40 KHz, and the homogenate was centrifuged for 10 min at 600 ϫ g to remove nuclei. The supernatant was centrifuged at 100,000 ϫ g for 1 h. The resulting membrane pellets were resuspended in homogenizing buffer, and aliquots of the total homogenate and membrane fractions were stored at Ϫ80°C. Protein was determined by the method of Bradford (45) with bovine serum albumin as a standard.
Protein Separation and Immunoblotting-Proteins were separated by SDS-PAGE (7.5% resolving gel for PLC and 13% for G-protein) in 25 mM Tris base, pH 8.3, 192 mM glycine, 0.1% SDS (46). They were electrophoretically transferred to nitrocellulose membranes (Immobilon P, Millipore, St. Quentin-en-Yvelines, France) in the same buffer with 20% ethanol for 2 h at 100 V (47). Nonspecific binding to nitrocellulose was prevented by incubating the membranes in 50 mM Tris-buffered saline (TBS) pH 7.5 containing 150 mM NaCl, 5% skimmed milk powder and 0.05% Tween-20 for 12 h at 4°C. The membranes were washed in TBS containing 0.1% Tween-20 and incubated overnight at 4°C with polyclonal rabbit antibodies against specific isoenzymes of PLC (PLC-␤1, PLC-␤2, PLC-␤3, and PLC-␤4) and specific G-proteins (G␣ q/11 , G␣ s ). The concentrations of PLC antibodies in TBS, 1.5% skimmed milk, 0.1% Tween 20 were as follows: 0.1 g/ml for PLC-␤1, 0.5 g/ml for PLC-␤2, 1 g/ml for PLC-␤3, and 0.5 g/ml for PLC-␤4. The concentrations of G␣ q/11 and G␣ s antibodies in the same buffer were 0.2 g/ml and 0.5 g/ml, respectively. The antibodies bound to the proteins on the nitrocellulose were detected using peroxidase-conjugated goat anti-rabbit IgG (1 mg/ml) (diluted 1/5000 in TBS, 1.5% skimmed milk, 0.1% Tween 20  (48), and assays were done essentially as described by Wu et al. (49). Diluted membranes (10 l, 5-10 g of protein) in 50 mM HEPES, pH 7.0, 0.5 mM EGTA, 2 mM EDTA, 0.6 mM pepstatin, 0.5 mM benzamidine, 0.1 mM leupeptin, 2 mM phenylmethylsulfonyl fluoride, 0.125 mM aprotinin, and 1 mM dithiothreitol, were added to 40 l of assay buffer (50 mM HEPES, pH 7.0, 100 mM NaCl, 5 mM MgCl 2 , 0.6 mM CaCl 2 , and 2 mM EGTA) plus 10 l of PIP 2 (10,000 -12,000 cpm of [ 3 H]PIP 2 ), and incubated on ice for 10 min. For the antibody inhibition assay, membranes were incubated for 2 h with 10 l of antibody, at the concentrations indicated, prior to adding the reaction mixture. For competition assay with the peptide control, 10 l of the antibody to be tested were mixed FIG. 6. Responses of OHS-4 cells treated with antibodies against PLC isoforms to CGRP. Cells were cultured, loaded with Fura2/AM, and incubated with PLC antibodies as described. The concentrations of anti-PLC antibodies were 1 g/ml for PLC-␤1, 10 g/ml for PLC-␤3, 5 g/ml for PLC-␤4, 10 g/ml for PLC-␥1, and 10 g/ml for PLC-␥2. Only anti-PLC-␤1 antibody blocked Ca 2ϩ mobilization induced by CGRP. In some experiments, CGRP was added 30 s after 2 mM EGTA. These results are representative of at least six coverslips for each experimental case.
with 2 l of serially diluted peptide control before adding to the membranes and incubation for 2 h. The reaction was started by adding GTP␥S with or without CGRP, followed by incubation at 37°C for 15 min. The reaction was stopped by adding 0.5 ml of chloroform/methanol/ HCl (40:20:0.5), mixing, and chilling on ice. Soluble inositol phosphates (indicating PIP 2 hydrolysis) were extracted by adding 150 l of chloroform and 200 l of 0.1 M HCl. Phases were separated by centrifugation, and 200 l of the upper aqueous phase were taken for liquid scintillation counting.
Statistical Analysis-The data were analyzed by one-way analysis of variance. Treatment pairs were compared by Dunnett's method. A value of n indicates the number of glass coverslips used for a specific experiment or the number of cultures.
Mechanisms of CGRP-induced Changes in Intracellular Calcium Concentration-1 nM CGRP was added 30 s after 2 mM EGTA, or 60 s after 1 M nifedipine or 1 M verapamil. Verapamil itself caused a small (5%) decrease in [Ca 2ϩ ] i , whereas nifedipine caused a small (5%) transient increase (43). EGTA, nifedipine, and verapamil reduced the magnitude (50 Ϯ 10%, mean Ϯ S.E., n ϭ 8, p Ͻ 0.001) of the transient peak induced by CGRP, and the plateau phase was completely abolished ( Fig.  2A; data shown only for EGTA and nifedipine). Cells were incubated for 3 min with 1 mM neomycin, with 3 M U-73122 or with 2 M U-73343, a closed analog of U-73122 but inactive agent (43), before adding 1 nM CGRP. Neomycin itself caused a small (5%) decrease in [Ca 2ϩ ] i (44), whereas U-73122 caused a small (5%) increase (43). Neomycin and U-73122 both abolished the transient peak, but not the plateau phase (Fig. 2B). U-73343 (0.3-5 M) had no effect on the intracellular calcium response to CGRP (data not shown).
The cells incubated for 16 h with 100 ng/ml PTX showed no change in the basal level of [Ca 2ϩ ] i or in the [Ca 2ϩ ] i response to 1 nM CGRP (Fig. 3).
Western Immunoblotting of the PLC and G-proteins-All the anti-PLC-␤ antibodies used in this study were raised against amino acid sequences in the carboxyl terminus of each PLC-␤. Western immunoblotting showed a 150-kDa immunoreactive band using the anti-PLC-␤1 antibody, a 158-kDa immunoreactive band using the anti-PL-␤3 antibody, and a 153-kDa immunoreactive band using the anti-PLC-␤4 antibody (Fig. 4). No immunoreactive reactive band for PLC-␤2 was found (Fig. 4). Immunoblots probed with the anti-G␣ q/11 antibody or the anti-G␣ s antibody showed a 42-kDa immunoreactive band (Fig. 5). A competitive Western blot with polyclonal PLC-␤1, PLC-␤3, and PLC-␤4 antibodies and the antigens against which they were raised showed that immunoreactivity was completely abolished when a 100-fold excess of antigen was included (data not shown). This also shows that each antibody reacted with the intended target isoform.
PLC Isoenzymes and G-proteins Involved in the Effects of CGRP on Intracellular Calcium-Treament of the cells with saponin for 5 min followed by incubation for 60 min with the anti-PLC antibody or anti-G-protein antibody in the absence of FIG. 7. Responses of OHS-4 cells treated with antibodies against G-proteins to CGRP. Cells were cultured, loaded with Fura2/AM, and incubated with G-protein antibodies as described. The concentrations of anti-Gprotein antibodies were 2 g/ml for G␣ q/ 11, 5 g/ml for G␣ s , 5 g/ml for G␣ i , and 5 g/ml for G␤ plus 5 g/ml for G␥. Only anti-G␣ q/11 antibody blocked Ca 2ϩ mobilization induced by CGRP. These results are representative of at least six coverslips for each experimental case. saponin did not affect the basal [Ca 2ϩ ] i . Nonimmune serum had no effect on basal [Ca 2ϩ ] i or on the [Ca 2ϩ ] i response to CGRP.
The CGRP-induced increase in [Ca 2ϩ ] i was reduced by anti-PLC-␤1 antibody, whereas antibodies to PLC-␤3 and PLC-␤4 had no effect (Fig. 6). The residual increase was due to Ca 2ϩ influx because it was totally blocked by incubating the cells with 2 mM EGTA. Anti-PLC␥1 and anti-PLC␥2 antibodies had no effect on the [Ca 2ϩ ] i response to CGRP (Fig. 6), as did anti-PLC-␤2 antibody (data not shown).
Polyclonal anti-PLC-␤1 antibody was incubated for 2 h with its corresponding antigen or with the antigens used to produce the other anti-PLC antibodies (antibody:antigen 1:10 or 1:100) before use. The inhibition of the CGRP-induced increase in [Ca 2ϩ ] i due to anti-PLC-␤1 antibody totally disappeared only when the anti-PLC-␤1 antibody was incubated with its own antigen, but not with the antigens used to raise the other anti-PLC antibodies ( Fig. 6; data shown only for the antigen corresponding to PLC-␤1).
The CGRP-induced transient peak in [Ca 2ϩ ] i was blocked by anti-G␣ q/11 antibody (Fig. 7). The residual increase was due to a Ca 2ϩ influx because this was totally blocked by incubating the cells with 2 mM EGTA (Fig. 7). The anti-G␣ i antibody (1 g/ml), which cross-reacts with G␣ i1 , G␣ i2 , and G␣ i3 , and anti-G␣ s antibodies did not alter the [Ca 2ϩ ] i response to CGRP (Fig.  7). Adding the anti-G␤ antibody, which reacts with G␤1, G␤2, G␤3, and G␤4, and the anti-G␥ antibody to the cell had no effect on the [Ca 2ϩ ] i response to CGRP (Fig. 7). PIP 2 Hydrolysis-Since testing the effect of GTP␥S on intracellular calcium needed permeabilized cells, and this made it impossible to keep the antibodies inside the cell, we used a cell-free membrane system to test the activity of the antibodies. OHS-4 membranes containing endogenous PLC and G subunits were mixed with phospholipid vesicles containing radioactive substrate ([ 3 H]PIP 2 ). Fig. 8A shows the effect of CGRP on the hydrolysis of PIP 2 in the absence of GTP␥S. The effect of GTP␥S in the absence and presence of 1 nM CGRP showed that GTP␥S was ineffective below 1 M (Fig. 8B). GDP␤S (100 M) inhibited the increase induced by 10 M GTP␥S, 10 M GTP␥S plus 1 nM CGRP, or 1 nM CGRP alone (data not shown). OHS-4 membranes were incubated with antibodies prior to stimulation with 100 M GTP␥S. All anti-PLC antibodies (␤1, ␤3, and ␤4), like anti-G␣ q/11 and anti-G␤␥ antibodies, inhibited the PIP 2 hydrolysis induced by GTP␥S (Fig. 8C), whereas anti-G␣ s and anti-G␣ i antibodies had no effect (data not shown). Membranes incubated with both the antibody and the blocking peptide, gave the same amount of [ 3 H]inositol 1,4,5-trisphosphate as obtained with preimmune serum (Fig. 8C). DISCUSSION We have shown that G␣ q/11 -protein coupled to phospholipase C-␤1 is involved in the signaling of CGRP in human bone cells, and that G␤␥ subunits have no effect. The data also indicate that Ca 2ϩ mobilization from the endoplamic reticulum following the increased formation of inositol 1,4,5-trisphosphate induced by CGRP is due to activation of a G␣ q/11 that is insensitive to pertussis toxin.
CGRP increases intracellular calcium concentration within 5 s via two mechanisms. It causes an influx of Ca 2ϩ from the extracellular milieu via voltage-gated calcium channels as indicated by the experiments with EGTA, nifedipine, and verapamil. This mechanism is different from that of rat osteoblastic UMR106 cells, in which depleting extracellular Ca 2ϩ induces an efflux of intracellular Ca 2ϩ involving K ATP channels (18). Activating the membrane K ATP channels with high concentrations of CGRP (100 nM) could inhibit transmembrane Ca 2ϩ uptake by hyperpolarizing the membrane potential, so reduc-ing the voltage-dependent Ca 2ϩ channel activity (53). CGRP causes the release of Ca 2ϩ from the endoplamic reticulum in human OHS-4 cells as it does in rat UMR106 cells (18). This seems to be a general mechanism of CGRP action.
The activation of phosphoinositide-specific phospholipase C-␤ upon stimulation of specific cell receptors results in the formation of second messengers, diacylglycrol, a direct activator of protein kinase C, and inositol 1,4,5-trisphosphate (50,51). The latter binds to receptors on the endoplasmic reticulum, causing a transient release of calcium from the endoplasmic reticulum. As activation of PLC-␤ is an early event in the signal transduction pathway resulting in a variety of cellular responses, we first identified the PLC-␤ isoforms in bone OHS-4 cells. We find several isoforms of PLC-␤, ␤1, ␤3, and ␤4 but no PLC-␤2, which is present in the rat brain (52) and in rat osteoblasts (44). As confluent rat osteoblasts do not possess PLC-␤4 (44), whereas this isoform is present in the brain (52), these isoenzymes may well have a tissue-and/or species-specific distribution.
The next step was to identify the PLC-␤ isoform involved in the effect of CGRP. This is the first report that PLC-␤1 is involved in mobilization of Ca 2ϩ from the endoplasmic reticulum by CGRP. Anti-PLC-␤1 antibody inhibits the CGRP-induced increase in [Ca 2ϩ ] i in much the same way as direct (U-73122) or indirect (neomycin) inhibitors of PLC. Anti-PLC antibody, like PLC inhibitors, blocks only the part of the increase in [Ca 2ϩ ] i that is due to Ca 2ϩ mobilization from the endoplasmic reticulum. The inhibition of the enzyme activity by anti-PLC-␤1 antibody is totally abolished in competition experiments, in which polyclonal PLC-␤1 antibody is incubated with the antigen against which it was raised, but not when using the antigen corresponding to other PLC-␤. This type of enzyme inhibition by selective antibodies against phosphoinositide-specific PLC has also been demonstrated in fresh bovine erythrocytes (54) and rat osteoblasts (44), suggesting that the antibody directed against an amino acid sequence at the carboxyl terminus of the PLC-␤ binds to a part of the enzyme which is critical for the geometry of the active site. Anti-PLC-␤1 antibody also inhibits the formation of inositol 1,4,5trisphoshate in a OHS-4 cell-free membrane system. Neither PLC-␤3 and PLC-␤4 are involved in CGRP signal transduction, although both PLC stimulate PIP 2 hydrolysis in the presence of GTP␥S. Similarly, PLC-␥1 and -␥2 take no part in the effects of CGRP as expected, because PLC-␥ are substrates for growth factor receptor protein-tyrosine kinases (55).
It is very likely that only PLC-␤ is involved in the action of CGRP, as only PLC-␤ types are regulated via heterotrimeric G-proteins in response to agonist binding to receptors (55,56). Receptor activation of PLC via G-proteins occurs by pertussis toxin-sensitive and toxin-insensitive signaling pathways (26).
The ␣ subunits of the G q/11 family are presumed to mediate the toxin-insensitive pathway, but the nature of the G-proteins mediating the toxin-sensitive pathway is less well understood (55)(56)(57)(58). This study shows that the PLC-␤1 involved in the action of CGRP is linked to a pertussis toxin-insensitive Gprotein, and this PTX-insensitive G-protein is a member of the G q family. These results are consistent with the fact that the G-proteins G q/11 are the most prominent G-protein activators in receptor-mediated regulation of the PLC-␤1 (29). Moreover, forskolin does not mimic the effects of CGRP on [Ca 2ϩ ] i , 2 but increases cAMP formation in OHS-4 cells (19). Anti-G␣ i and anti-G␣ s antibodies do not block the [Ca 2ϩ ] i response to CGRP or the stimulation of PIP2 hydrolysis induced by GTP␥S. G␤␥ subunits, like PLC-␤3, which is the preferred target effector for G␤␥ subunits (36), are not involved in Ca 2ϩ mobilization induced by CGRP. These data indicate that the effects of CGRP on intracellular calcium in OHS-4 cells are not mediated via the G-protein linked to the cAMP pathway.
Activation of PLC via G-protein-coupled receptors requires higher ligand concentrations than for receptor-mediated adenylyl cyclase activation (22). But, we find that PLC is activated in OHS-4 cells at concentrations similar to those that increase cAMP in other systems.
Although the main second messenger produced in response to CGRP remains cAMP in various cell types, probably via G␣ s -protein, CGRP acts via the phospholipase C pathway and G␣ q/11 protein in human bone cells. The present data therefore strongly suggest that the dual signaling of CGRP is due to the activation of more than one G-protein, G␣ s for the cAMP pathway and G␣ q/11 for the PLC pathway (Fig. 9).
FIG. 9. Signals generated by CGRP. A, diagram showing the proposed coupling of CGRP receptors to phospholipase C and adenylyl cyclase via pathway 1 (two different G-proteins) or via pathway 2 (one G-protein acting through its ␣ and ␤␥ subunits). B, signaling pathway used by CGRP in human OHS-4 bone cells. CGRP uses pathway 1 since it activates phospholipase C-␤1 via a pertussis-insensitive G␣ q/11 protein, the ␤␥ subunits being ineffective.