0013-7227/92/1301-0029$03.00/0 Endocrinology Copyright 0 1992 by The Endocrine

Vol. Printed

Society

130, No. 1 in U.S.A.

Structure-Function Relationship of Parathyroid Hormone: Activation of Phospholipase-C, Protein Kinase-A and -C in Osteosarcoma Cells* AKIRA

FUJIMORI,

SU-LI

CHENG,

LOUIS

V. AVIOLI,

Division of Bone and Mineral Metabolism, Jewish Hospital Washington University Medical Center, St. Louis, Missouri

AND ROBERTO

CIVITELLI

St. Louis at 63110

of

ABSTRACT. Recent evidence indicates that after PTH interaction with its receptor, both protein kinase-A (PKA) and protein kinase-C (PKC) are activated. To investigate the relationship between PTH structure and protein kinase stimulation, we have analyzed the effects of synthetic PTH fragments on PKA and PKC in the rat osteogenic sarcoma cells, HMR 10601. Activation of PKA bv lo-’ M bovine (b) PTH-(l-34) was maximal (2.7-fold of control) at 5 min and remained elevated 15 min after hormone exposure. bPTH-(2-34), at equimolar doses, also stimulated PKA, but with a lower potency (1.4-fold of control), whereas propionyl bPTH-(2-34) [pbPTH-(2-34)], bPTH-(3-34), [Ty?]bPTH-(7-34) amide [bPTH-(7-34)], and bPTH-(30-34) were ineffective. On the other hand, translocation of PKC activity from the cytosol to the membrane after exposure to bPTH-(1-34) was transient, with a peak at 1 min (1.9-fold of control), and returned to basal levels after 5 min.

Other fragments, bPTH-(2-34), pbPTH-(2-34), bPTH-(3-34), and bPTH-(7-34), were also active on PKC, with relative ootenties of 81%, 67%, 62%, and 51% of bPTH:(l-34), respectively, whereas bPTH-(30-34) was inactive. bPTH-(l-34). bPTH-(234), pbPTH-(2-34), and bPTH-(3-34) also’ induced inositol 1,4,5-trisphosphate production, with a potency order of 1.6-, 1.6-, 1.5-, and 1.6-fold over the control value, respectively, thus indicating activation of phospholipase-C. Neither bPTH-(7-34) nor bPTH-(30-34) caused a statistically significant increase in inositol 1,4,5-trisphosphate production. These results demonstrate that PTH signal transduction through the two different pathways can be dissociated; while activation of the cAMP/PKA system requires amino acids 1 and 2, the phospholipase-C/PKC system is coupled to a longer domain of the hormone’s Nterminus. (Endocrinology 130: 29-36, 1992)

0

STEOBLASTS possess PTH receptors, which mediate PTH actions in bone tissue (l-3). The transduction of PTH signal through plasma membrane involves both stimulation of adenylate cyclase, with CAMP production (4, 5), and phospholipase-C-dependent hydrolysis of membrane-associated phosphatidyl inositol 4,5-bisphosphate (PInsPz), leading to generation of inositol 1,4,5trisphosphate (InsPs) and diacylglycerol (DAG) (6). After second messenger production, the event cascade of signal transduction continues through different pathways; while CAMP activates protein kinase-A (PKA) (7), InsPB releases calcium (Ca”‘) from intracellular stores (B), and DAG causes translocation of protein

kinase-C (PKC) from the cytosol to the cell membrane (9, 10). PKC, which requires Ca2+ and phospholipids for activation, relays information of various extracellular signals across the cell membrane (11, 12). Although all of these intracellular biochemical steps have been demonstrated to occur after PTH receptor binding, selective activation of single pathways has not been well established. This issue is relevant to osteoblast function if one considers that the physiological effects of the CAMP/ PKA and Ca2+/PKC systems are not always parallel. For example, we have previously reported that the two systems act synergistically to stimulate collagenase production in the UMR 106-01 osteogenic sarcoma cell line (13). On the contrary, while the antimitogenic action of PTH in this cell line is mediated by cAMP/PKA, activation of PKC leads to cell proliferation (14, 15). The selective control of each signal transducing pathway could bear potentially important implications for the design of PTH analogs with selective anabolic activity. For most biological assays, including CAMP production, the first 34 amino acids of PTH retain the full biological activity of the intact hormone, and truncation at the N-terminus causes a progressive loss of hormonal

Received July 24, 1991. Address all correspondence to: Akira Fujimori, M.D., Division of Endocrinology- and Bone Metabolism, Jewish Hospital of St. Louis, 216 South Kingshighway, St. Louis, Missouri 63110: Address requests for reprints to: Roberto Civitelli, M.D., Division of Endocrinology and Bone Metabolism, Jewish Hospital of St. Louis, 216 South Kingshighway, St. Louis, Missouri 63110. * This work was sunnorted in Dart bv NIH Grant AR-32087. NIH Training Grant AR-07633, and grants from the Shriners Hospital for Crippled Children. Part of this work has been presented at the Annual Meeting of the American Society for Bone and Mineral Research, Atlanta, GA, August 27-31, 1990 (Abstract 603). 29

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30

PTH FRAGMENTS

AND PROTEIN

activity (16-18). We have recently reported that the 334 fragment of bovine (b) PTH [bPTH-(3-34)], which did not induce CAMP synthesis in the osteogenic sarcoma cell line UMR 106-01, was, nevertheless, able to transiently increase the intracellular calcium concentration ([Ca”]J (18). This finding leads to the hypothesis that bPTH-(3-34) may also activate PKC, via stimulation of phospholipase-C, in the absence of PKA activation. In the present study we have examined the effects of PTH fragments shortened at the N-terminus on both PKA and PKC in the osteosarcoma cell line UMR 10601. We also measured InsPs production to verify whether PKC activation by PTH is associated with phospholipase-C hydrolysis of PInsPa. We found that, like the second messengers CAMP and [Ca2+]i (18), the two protein kinases can be differentially activated by some PTH fragments in UMR 106-01 cells. Materials

and Methods

Reagents and chemicals

Synthetic bPTH-(1-34) and bPTH-(3-34) were purchased from Bachem,Inc. (Torrance, CA). [Tyr34]bPTH-(7-34) amide [bPTH-(7-34)] was the kind gift of Drs. Michael Rosenblatt and Michael P. Caulfield (Merk, Sharp, and Dohme Research Laboratories, West Point, PA). bPTH-(Z-34), propionyl bPTH-(2-34) [pbPTH-(2-34)], and bPTH-(30-34) were generously provided by Dr. Kam F. Fok (Monsanto Co., St. Louis, MO). The chemical compositions of these fragments were confirmed by amino acid analysisand massspectrometry. PTH peptides were dissolvedin 1 mM HCl and stored at -20 C. [y-“‘P]ATP was obtained from AmershamCorp. (Arlington Heights, IL). Myo-[2-“Hlinositol was purchasedfrom DuPont (Billerica, MA); [3H]Ins(1,4,5)P3 and a mixture of [3H]InsP, [“H]InsP,, and [3H]InsP3 was obtained from Amersham. All other chemicalsand the tissue culture media were obtained from Sigma Chemical Co. (St. Louis, MO). UMR 106-01 cells were the generousgift of Dr. Nicola C. Partridge (St. Louis University, St. Louis, MO). Cell cultures

In these studiesthe clonal cell line UMR 106-01 was used between passages16 and 22. These cells are derived from the rat osteogenicsarcoma cell line UMR 106, which has been characterized as having an osteoblasticphenotype (5, 19, 20). They were maintained asdescribedpreviously (13, 18, 21). Measurement

of PKA activity

PKA activity was measuredaccording to the method of Partridge et al. (5, 7). In brief, confluent UMR 106-01 cells grown in 35-mm plastic dishes were washedwith serum-free Eagle’sMinimal Essential Medium with Earle’s salts and incubated at room temperature with PTH fragments in the presenceof 0.5 mM isobutylmethylxanthine (IBMX) for 1-15 min. The cellswerewashedthree timeswith ice-cold Dulbecco’s PBS and harvested in microfuge tubes by scraping and brief centrifugation. The cells were lysed by vortexing in 50 ~1

KINASES

Endo. 1992 Vol 130. No 1

extraction buffer [50 mM KH2P04, 10 mM Na2EGTA, 10 mM Na2EDTA, 2 mM IBMX, 2% Triton X-100,0.2 mM dithipthreitol (DTT), and 20 mM NaF, pH 7.21.The tubes were centrifuged, and the supernatant was collected. PKA activity was determinedby incorporation of [-r-32P]ATP into Kemptide, the synthetic peptide substrate for PKA (22). Ten microliters of the supernatant were incubated for 5 min at 25 C in 20 mM 2(N-morpholino)ethanesulfonic acid (MES) buffer (pH 6.8) containing 10 mM Mg(CH&00)2, 1 mM Na2EDTA, 0.1% BSA, 156 pg Kemptide, and 0.125 mM [-r-32P]ATP (400-1400cpm/ pmol) with or without 6.25 pM CAMP in a total volume of 80 ~1. At the end of the incubation, 25 ~1 of the assaymixture werespotted on a 1.5 x 1.5-cmWhatman P81 phosphocellulose paper (Whatman, Clifton, NJ), which was washedwith 10% acetic acid. The papers were transferred to scintillation vials, air dried, and counted using scintillation fluid. Hormonal activation of PKA was expressedas the PKA activity ratio, the ratio of the PKA activity measuredin the absenceof added CAMP to that obtained in the presenceof a saturating concentration of CAMP (-cAMP/+cAMP). Measurement

of PKC activity

These experiments were carried out using a modification of the method of Abou-Samra et al. (9). UMR 106-01 cells were grown in loo-mm plastic dishes. At confluence, cells were exposedto PTH fragments or phorbol 12-myristate 13 acetate (PMA) for 1-15 min. The cells were then washedthree times with ice-cold PBS and harvested by scraping and centrifugation. The cells were resuspendedin 4 ml ice-cold buffer A (20 mM Tris-HCl, 2 mM EDTA, 0.5 mM EGTA, 2 mM DTT, 0.001% leupeptin, and 0.5 mM phenylmethylsulfonylfluoride, pH 7.4) and homogenizedon ice with a tightly fitted Potter-Elvehjem tissue grinder (Wheaton, Millville, NJ). The homogenatewas centrifuged at 100,000X g for 45 min. The supernatant containing the cytosol was collected and stored in an ice bath. The pellet containing the membranewas resuspendedin buffer A supplementedwith 0.5% Nonidet P-40, agitated on ice for 60 min, and then centrifuged at 100,000 X g for 45 min. The supernatant with solubilized membraneswas collected. Both cytosol and membranefractions were applied to DEAE-cellulose columns (1 x 1 cm) that had been preequilibrated with buffer A. Since preliminary experiments revealedthat most of the PKC activity waseluted in 80 mM NaCl, the columnswere washedwith buffer A, and then PKC-enriched fractions were eluted with 2 ml buffer A containing 80 mM NaCl. PKC activity was determined by incorporation of [-r-32P]ATP into histone 111s.Twenty microliters of PKC-enriched fractions were incubated in an assaymixture containing (final concentrations) 20 mM Tris-HCl (pH 7.4), 5 mM Mg(CH&00)2, 2 mM DTT, 1 mM CaC12,125 pg/ml phosphatidylserine, 3 pg/ml 1,2-diolein, 250pg/ml histone-IIIS, 10 pM ATP, and 2 &i/ml [T-~‘P]ATP in a total volume of 80 ~1.The incubation wascarried out at 25 C for 5 min and terminated by transfer of the assaymixture onto 1.5 x 1.5-cmWhatman P81 phosphocellulosepaper,which was immediately washed with 75 mM phosphoric acid. The filter paper was transferred to a scintillation vial and counted using scintillation fluid. The protein content of each sample wasdeterminedusing the Bio-Rad Protein Assay Kit (Bio-Rad Laboratories, Richmond, CA). PKC activity was calculated

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PTH FRAGMENTS

AND PROTEIN

KINASES

31

from the difference in phosphorylation, assayed in the presence and absence of Ca’+, into histone-111s per pg protein, and the hormonal effect as the PKC translocation ratio, the ratio of enzyme activity in the membrane to that in the cytosol. Measurement of InsP3 The method describedpreviously (23) was used. In brief,

UMR 106-01 cells were grown in 25cm2 plastic flasks (Costar, Cambridge,MA) to confluence and labeled with myo-[2-3H] inositol (1.5 &X/ml). The cells were exposedto either PTH fragments or vehicle (1 mM HCl) for 30 set, and the reaction wasterminated by mediumremoval and immediateaddition of 1% trichloroacetic acid. This incubation time was chosen on the basisof previousdata indicating that PTH effect on inositol phospholipid metabolism is maximal after 30 set of hormone exposure (6). The flasks were left at 4 C for 30 min to allow extraction of the water-solubleinositol phosphates.The acidic extracts were neutralized with saturated KHCOs and applied to solid phase extraction tubes containing aminopropyl absorbent (Supelclean, Supelco, Belfefonte, PA). The tubes were washedand eluted with 0.5 ml 1.5 M NH,OH. Inositol poly-

2 0.0 0

1

5 Time

15 (min)

FIG. 1. Time course of the effect of 10m7 M hPTH-(1-34) on PKA activity in UMR 106-01 cells. Cells were incubated with bPTH-(1-34) for O-15 min, and PKA activity was measured, as described in Materials and Methods. Results are representative of three separate experiments performed in triplicate. Missing SE bars are contained within the symbols. *, P < 0.05 compared with basal value. 1.2 E 3

* 1.0 T

phosphates were separated on HPLC Dionex Ion Pat A55A 5pm (Sunnyvale, CA) ion exchange column. A three-step gradient of 92:8, 48~52,and 25:75 (vol/vol, water-150 mM NaOH), with a flow rate of 1 ml/min, was used to elute the inositol phosphates.The elution position of each metabolite (InsP,

z z Y 2

InsP,, and InsPB) was determined using radiolabeled standards. Statistical analysis

0.2 -0.0

Results are expressedas the mean f SEM unlessotherwise indicated. All data illustrated are representative of at least three experiments performed under the same conditions. Analysis of variance and Duncan’s multiple range test were used to assessthe differences between multiple experimental groups.

Results Activation

0.4~-

of PKA

Figure 1 shows the time course of the effect of 10m7 M bPTH-(1-34) on PKA activity in UMR 106-01 cells. After 1-min incubation with bPTH-(l-34), the PKA activity ratio significantly increased. Maximal activation (2.4-fold above control) was observed at 5 min. The enzyme activity remained almost maximal 15 min after hormone addition. Similar results were obtained in the absence of IBMX (not shown). Consistent with our previous study on CAMP production (18), only bPTH-(l34) and bPTH-(2-34), at equimolar doses, were active on PKA. The stimulatory activities of the two peptides were 2.7 + 0.03 and 1.4 f 0.03-fold over the control value, respectively (Fig. 2). On the other hand, pbPTH-(2-34), which is identical to bPTH-(1-34) except for the lack of the amino group of alanine in position 1 (18), as well as the other shorter fragments, bPTH-(3-34), bPTH-(734), and bPTH-(30-34), failed to activate PKA (Fig. 2).

FIG. 2. Effects of 10m7 M PTH fragments on PKA activity in UMR 106-01 cells. Cells were incubated with PTH fragments for 5 min, and PKA activity was determined, as described in Materials and Methods. Results are representative of four separate experiments performed in triplicate. *, P < 0.05 compared with control.

Activation

of PKC

To validate our PKC methodology, we first tested the effect of lop7 M PMA, a direct activator of PKC (12). After cell exposure to PMA, there was a sustained timedependent increase in membrane-associated PKC activity, with a concomitant decrease in cytosolic PKC activity and a consequent increase in the PKC translocation ratio at all time points (Fig. 3). On the other hand, the effect of 10m7 M bPTH-(l-34) was transient, with a peak at 1 min and a return to basal levels after 5 min (Fig. 4). In addition, the amplitude of the PTH effect was lower than that of PMA; the PMA/bPTH-( l-34) potency ratio at 1 min was 1.5 f 0.1. In contrast to what was observed for PKA, other PTH fragments were active on PKC. As shown in Fig. 5, equimolar dose (10m7 M) of bPTH-(234) and pbPTH-( 2-34) also caused translocation of PKC activity.

Figure 6 shows the effect of further deletion of

the N-terminus. Both bPTH-(3-34) and bPTH-(7-34) caused PKC translocation, although the effect of the

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32

PTH

-I

0

1

** L-L 5

lime

FRAGMENTS

AND

PROTEIN

KINASES

Endo. 1992 Voll30. No 1

* =mm&a” l

bPT”-(2-54) 15

(min)

pbiI’W2-34)

bPlH-(l-34)

5. Effects of lo-’ M bPTH-(l-34), bPTH-(2-34), and pbPTH-(234) on PKC activity in UMR 106-01 cells. After 1-min incubation with PTH fragments, PKC activity was determined in both membrane and cytosol fractions, as described in Materials and Methods (lower panel). Results are also expressed as the PKC translocation ratio (upperpanel) and are representative of three separate experiments performed in triplicate. *, P < 0.05 compared with control. FIG.

FIG. 3. Time cokse of the effect of lo-’ M PMA on PKC activity in UMR 106-01 cells. Cells were incubated with PMA for O-15 min. Both membrane-bound and cytosolic PKC activities were determined (lower panel). The change in the PKC translocation ratio is shown in the upper panel. Results are representative of three separate experiments performed in triplicate. Missing SE bars are contained within the symbols. *, P c 0.05 compared with basal value.

l

0

1

5

15

Control

Time (min)

bPTH-(7-34)

bPTH-(3-34) b&W-(1-34)

4. Time course of the effect of lo-’ M bPTH-(l-34) on PKC activity in UMR 106-01 cells. Cells were incubated with bPTH-(l-34) for O-15 min. Both membrane-bound and cytosolic PKC activities were determined (lower panel). The change in the PKC translocation ratio is shown in the upperpanel. Results are representative of three separate experiments performed in triplicate. Missing SE bars are contained within the symbols. *, P < 0.05 compared with basal value.

6. Effects of 10e7 M bPTH-(l-34), bPTH-(3-34), and bPTH-(734) on PKC activity in UMR 106-01 cells. After 1-min incubation with PTH fragments, PKC activity was determined in both membrane and cytosol fractions, as described in Materials and Methods (lower panel). Results are also expressed as the PKC translocation ratio (upperpanel) and are representative of three separate experiments performed in triplicate. *, P < 0.05 compared with control.

latter did not reach statistical significance by multiple range test. In agreement with our previous data on [Ca2+]i (18), the potency of these fragments decreased with stepwise shortening of the N-terminus. The relative potencies of the shorter PTH fragments are shown in

Table 1. On the contrary, bPTH-(30-34), which had been shown to be the core domain for a mitogenic effect of PTH on chicken chondrocytes (24) was inactive in our PKC assay (n = 3).

FIG.

FIG.

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PTH TABLE 1. Relative potencies of PTH fragments InsPB in UMR 106-01 osteogenic sarcoma cells

100 23 -2 -4 -5 -5

on protein

PKC

PKA bPTH-(l-34) bPTH-(2-34) pbPTH-(2-34) bPTH-(3-34) bPTH-(7-34) bPTH-(30-34)

FRAGMENTS

+ + + + f +

2 2 5” 2” 2” 2”

100 81 67 62 51 12

f + + + + +

kinases

AND and

InsPa 17 6 12 17 17” 9”

100 + 99 + 70 + 88k8 43 + 18 +

11 23 13 10” 6”

Measurements of PKA, PKC, and InsPI were performed as described in Materials and Methods. Data represent the effect of an equimolar (10-r M) concentration of all peptides and are normalized to the average effect of bPTH-(l-34) in order to compare potencies. Results are representative of at least three similar experiments for each assay. ’ Not significantly different from control.

zz3=‘7‘3 1 7 g;NN!?c: I I $ g

7 fj a

; ’ g

: :$’

; sI E

FIG. 7. Effects of 10-r M PTH fragments on InsPB production in UMR 106-01 cells. Cells prelabeled with myo-[3H]inositol were incubated with PTH fragments for 30 sec. InsPa in the cell extract was separated using HPLC, as described in Materials and Methods. Data are expressed as lo3 counts per min, corrected for protein content, and are representative of three separate experiments. *, P < 0.05 compared with control.

Stimulation of InsP3 production

To verify whether the action of PTH fragments on PKC is associated with activation of phospholipase-C, the effect of PTH fragments on InsPB production was also studied. In agreement with our previous results (6), cell exposure to lo-’ M bPTH-(1-34) was followed by a rapid production of InsPs (Fig. 7). bPTH-(2-34), pbPTH-(2-34), and bPTH-(3-34) also stimulated InsPB production. While bPTH-(2-34) was almost as effective as bPTH-(l-34), the potency of the other fragments decreased with progressive deletion of the amino acids at the N-terminus, as observed for the PKC assay. Although bPTH-(7-34) increased InsPB production by 1.3fold over the control value, the difference was not statistically significant. Again, bPTH-(30-34) did not elicit any effect on InsPB production. Table 1 summarizes the potencies of all PTH fragments tested in this study relative to the activity of bPTH-(1-34).

PROTEIN

KINASES

33

Discussion The present study demonstrates that pbPTH-(2-34), bPTH-(3-34), and bPTH-(7-34), which are unable to activate PKA, have agonist effects on PKC in UMR 10601 cells, suggesting that while activation of the CAMP/ PKA system requires amino acids 1 and 2, the phospholipase C/PKC system is coupled to a longer domain of PTH N-terminus. PKC, which is present in an inactive form in soluble cytosol or loosely bound to the membrane under resting conditions, is activated by tight association with the membrane in a signal-dependent manner (11, 25, 26). The effect of PTH fragments on PKC was transient, with a peak at 1 min and a return to baseline after 5 min of hormone exposure. This response pattern is consistent with other reports on UMR 106-01 and ROS 17/2.8 osteosarcoma cells (9, 10) as well as opossum kidney (OK) cells (27). The time course of the effect of PTH on PKC parallels the hormone’s effect on [Ca2+]i (8, 18, 21) and InsPs production (6). This strict temporal correlation is compatible with the hypothesis that all of these biochemical steps are consequent to stimulation of membrane-bound phospholipase-C and consequent initiation of the signal cascade leading to production of InsPB and DAG and activation of PKC. In keeping with this hypothesis, G-protein-dependent activation of phospholipase-C has been reported by Babich et al. (28) in the cell line used in this study. The different time courses of PTH peptides and PMA on PKC translocation might be explained by the much slower catabolism of the phorbol ester. Its stability is able to deliver a stronger and more sustained stimulatory signal to the enzyme than the natural DAG, generated through hormone-induced hydrolysis of PInsP2 (12). Moreover, the affinity of PMA binding to the PKC molecule is higher than that of DAG (29). On the other hand, the effect of PTH on PKA was more prolonged, a finding that is in line with the experience of others (7, 27). Therefore, if PKC-dependent physiological effects are strictly linked to PKC translocation, long term PTH signaling appears to be mainly mediated through the cAMP/PKA pathway. The significance of transient and sustained intracellular signaling in PTH and osteoblast physiology requires additional investigation. Martin et al. (30) have reported a weak agonist activity of [Nles,Nle1s,Tyr34]bPTH-(3-34) amide on PKA in OK cells. This modified 3-34 fragment of PTH was inactive on CAMP production, but slightly enhanced PKA activity at high concentrations ( lop6 M), leading to the conclusion that PKA might be a more sensitive indicator of CAMP/ PKA system activation than total cellular CAMP. In the present study we could not detect any agonist activity of

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34

PTH FRAGMENTS

AND PROTEIN

the native 3-34 peptide, which was also inactive on CAMP, even after enhancement of the CAMP response by cell preincubation with pertussis toxin (18). The lofold lower dose of peptide we used in this study might explain the apparent discrepancy. Alternatively, the higher chemical stability of [Nle8,Nle’8,Tyr34]bPTH-(334) amide compared to that of the native peptide may result in an apparent higher potency (31). Although unlikely, the possibility that the activity of 3-34 fragments on the cAMP/PKA system may be different in osteoblasts and renal cells because of different receptor affinities cannot be discounted. Finally, species differences (rats US. opossum) could be responsible for the discrepancy. In any case, the present results clearly establish that at a concentration of 10e7 M, native bPTH(3-34) does not activate the cAMP/PKA second messenger system in rat osteoblastic cells. The 3-34 fragment was initially identified as the first antagonist of PTH action (30, 31). Subsequently, however, a weak but consistent agonist activity of this fragment was demonstrated in uiuo (32-34). In addition to its activity on InsP3 and PKC observed in the present study, recent reports from our and other laboratories have shown that bPTH-(3-34) is active on [Ca2+]i (18, 35,36). It is, therefore, plausible that some of the in uiuo agonistic effects of this peptide may be mediated by the [Ca2+]i/PKC signal transducing pathway. On the other hand, bPTH-(7-34) is devoid of PTHlike actions on both bone and kidney in uiuo (37, 38). Nevertheless, bPTH-(7-34) showed a detectable, albeit weak, agonist activity in both the PKC and IP3 assays. It is possible that the effect of this fragment on phospholipase-C may be at the limit of sensitivity of our assays, thus accounting for our inability to demonstrate statistically significant increases in InsP3 and PKC translocation. A potential agonist effect of bPTH-(7-34) on the phospholipase-C/PKC system would be in contrast with our previous study, in which bPTH-(7-34) consistently failed to increase [Ca2+]i in the same cell line (18). Donahue et al. (36) did observe positive effects of bPTH-(7-34) on [Ca2+]i in ROS 17/2.8 cells, but only at concentrations of 10e6 M and higher. The peptide was virtually ineffective on [Ca2+]i at the dose used in our study (low7 M). To our knowledge, there are no other examples of hormones or factors active on inositol phosphate metabolism but without effects on [Ca2+]i. Although a more in-depth analysis of the mechanisms of bPTH-(7-34) interaction with the PTH receptor should provide some insights, a few hypotheses can be put forward to explain these puzzling results. First, if indeed the effect of PTH on [Ca2+li requires both intracellular Ca2+ release and opening of a membrane Ca2+ channel, as hypothesized by Yamaguchi et al. (39), then the lack of effect of the 7-34 fragment on [Ca2+]i might be due to

KINASES

Endo. Voll30.

1992 No 1

the peptide’s inability to operate the membrane-bound mechanism of Ca2+ influx. Alternatively, if generation of a [Ca2+]i transient requires a threshold level of InsP3, then one might speculate that the shortened PTH fragment can only release subthreshold amounts of the second messenger, inadequate to trigger a [Ca2+]i transient. This hypothesis seems to be supported by the effect of pbPTH-(2-34), which, although less potent than bPTH(l-34) and bPTH-(2-34), did induce [Ca2+]i transients (18) and produce significant amount of IP3 (this study). Since PKC and [Ca2+]i work synergistically to evoke cellular responses (12), one consequence of the peculiar effect of bPTH-(7-34) on the [Ca2+]i/PKC system might be a blunted or ineffective progression of the hormonal signal through the subsequent biochemical steps in the absence of a simultaneous rise in [Ca2+]i and PKC translocation. This incomplete signal transduction may be the basis of the lack of agonist properties of bPTH-(7-34) in uiuo. Figure 8 summarizes in a graphic fashion our accumulated data on PTH structure and signal transduction. Some of the biochemical steps illustrated are still hypothetical. According to this model, activation of both cAMP/PKA and [Ca2+]i/PKC systems occurs through a single receptor, and the signal is split into the different pathways by multiple G-proteins. Activation of adrnylate cyclase requires not only binding of the hormone to its receptor, but also direct interaction of the N-terminal tail of the peptide with a specific domain of the receptor.

CAMP

FIG. 8. Schematic illustration of a model for PTH signal transduction. Steps that are still hypothetical are indicated by a question mark. The first two amino acids of the N-terminus are coupled to adenylate cyclase through stimulatory (G.) and inhibitory (G,) GTP-binding proteins. Activation of adenylate cyclase leads to CAMP production and stimulation of PKA, which dissociates into its regulatory (R) and catalytic (C) subunits. Amino acids 3-7 (or longer) are coupled to phospholipaseC, probably via a different GTP-binding protein, tentatively indicated as G,. Phospholipase-C catalyzes the hydrolysis of PInsP:, into DAG and InsPB. DAG, in turn, activates PKC, whereas InsPs mobilizes Ca*+ from intracellular stores. Increased cytosolic free Ca2+ activates calmodulin and synergizes with PKC.

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PTH

FRAGMENTS

AND

Under this condition, phospholipase-C is also fully activated. The latter effector enzyme can also be activated by interaction of a longer domain, located down-stream to the first two amino acids, with the receptor molecule, thus allowing signal transduction through the [Ca”]i/ PKC pathway to occur in the absence of adenylate cyclase activation. Our studies on the structure-function relationship of PTH has revealed that the fragments lacking at least the first two, but not more than the first six, amino acids of the PTH native sequence are inactive on the CAMP/ PKA system, but retain agonist properties on the [Ca2+]i/PKC system. We believe that these shortened PTH peptides may represent useful tools not only to dissect the physiological roles of different second messenger systems, but also in the development of pharmacologically active compounds. In uiuo studies have, in fact, demonstrated that l-34 fragments of PTH, when used at low doses, gradually increase bone formation, possibly through direct stimulation of osteoblasts (4042). Since the [Ca2+]i/PKC system stimulates cell proliferation (12, 14, 15), shortened PTH fragments may display a prevalent anabolic action on bone metabolism compared to the l-34 peptide. Acknowledgments

We wish to thank Marilyn Roberts for preparation of the tissue cultures. We also wish to thank Drs. Michael Rosenblatt, Michael P. Caulfield, and Kam F. Fok for PTH fragments, and Dr. Nicola

C. Partridge

for UMR

106-01 clonal cell line.

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