INTRODUCTION

Parathyroid hormone (PTH),¹ PTH-related peptide (PTHRP), and their N-terminal analogues influence plasma calcium and phosphate homeostasis by regulating a variety of membrane ion channels and transporters within target cells, including Ca²⁺ channels (1), Cl channels (2), Na⁺/P_i cotransporters (3, 4), and Na⁺/H⁺ exchangers (5, 6, 7, 8). This multiplicity of actions is generally believed to reflect ligand binding to a common heterotrimeric G-protein-coupled receptor that is linked to multiple effector systems (i.e. adenylate cyclase and phospholipase C) (9). This is exemplified in renal proximal tubule OK cells where N-terminal peptide fragments of PTH and PTHRP (i.e. PTH-(1-34), PTH-(3-34), PTH-(28-42), PTH-(28-48), and PTHRP-(1-34)) rapidly inhibit the activity of the apically localized Na⁺/H⁺ exchanger NHE3 isoform by a mechanism involving cAMP-dependent protein kinase (PKA) and Ca²⁺/phospholipid-dependent protein kinase (PKC) (7). This regulation is remarkable given that PTH (an 84-amino acid peptide) and PTHRP (a 139-173-amino acid peptide depending on the species) (10) share minimal identity in primary structure, with only 8 of the 13 N-terminal amino acids being common between these two peptides. PTH and PTHRP require the first two N-terminal amino acids and amino acids 25-34 to stimulate adenylate cyclase activity (11, 12, 13, 14). In contrast, amino acids 3-34 and even smaller regions (amino acids 28-34) appear sufficient to activate PKC translocation to the plasma membrane (15, 16, 17).

However, the universality of this signaling paradigm to account for the diverse actions of N-terminal analogues of PTH and PTHRP is no longer tenable in view of recent data reporting the existence of additional related receptors. Usdin et al. (18) recently isolated and characterized a unique receptor (called PTH2) from a rat brain cDNA library that shares 70% amino acid identity to the classical PTH/PTHRP receptor (type I). PTH2 mRNA is expressed predominantly in brain and pancreas, and to a much lesser extent in placenta and testis. PTH2 is also functionally distinguished from the type I receptor by its selective binding of PTH and by its potent activation of adenylate cyclase activity.

In addition, biochemical studies in normal keratinocytes and squamous carcinoma cell lines suggest the existence of a novel PTH/PTHRP receptor (type II) that differs qualitatively in its intracellular signaling properties from those of the type I receptor and PTH2 (19). This potential receptor is activated by N-terminal peptide fragments of both PTH and PTHRP, leading to increases in intracellular Ca²⁺ but not cAMP. These cells also express multiple mRNA transcripts that hybridize to type I receptor cDNA probes, yet differ significantly in size from the type I receptor mRNA present in human bone SaOS-2 cells. Differently sized transcripts are also observed in rat kidney, liver, skin, and testes (20). These data have been interpreted to indicate the presence of a distinct gene product or an alternatively spliced variant of the type I receptor.

Other circumstantial evidence also supports the existence of multiple receptors. A C-terminal peptide of human PTH (i.e. PTH-(53-84)) elicits a number of biological responses in rat osteosarcoma cells (21, 22) yet fails to bind to the human PTH/PTHRP receptor type I stably expressed in human embryonic kidney (HEK-293) cells (23). Thus, it is possible that the biological activity of some of these N- and C-terminal analogues is actually elicited by selective binding to other, as yet uncharacterized, PTH/PTHRP receptors. Understanding the signaling mechanism of these PTH/PTHRP analogues is of physiological relevance as parathyroid cells normally secrete peptide fragments of PTH (24).

In view of these data, we tested the hypothesis that N-terminal analogues of PTH and PTHRP can bind to and activate the PTH/PTHRP receptor type I, stimulate PKA and PKC, and acutely regulate the activity of the Na⁺/H⁺ exchanger NHE3 isoform, as is believed to occur in renal cells. This was accomplished by transient and stable transfection of cDNAs encoding the rat PTH/PTHRP receptor type I and the rat Na⁺/H⁺ exchanger NHE3 isoform into Chinese hamster ovary AP-1 cells that are devoid of endogenous Na⁺/H⁺ exchanger activity and lack responsiveness to PTH or PTHRP. The data clearly demonstrate that the AP-1-transfected cells were able to bind the various PTH and PTHRP analogues, stimulate production of multiple second messengers, and elicit biological responses in a manner that precisely mimics the responses observed in renal OK cells. Although the data do not exclude the presence of other PTH/PTHRP receptors in OK cells, it suggests that the PTH/PTHRP receptor type I is sufficient to mediate the diverse biological actions of these N-terminal PTH and PTHRP analogues in these cells.

EXPERIMENTAL PROCEDURES

Phosphatidyl-L-serine, diolein, dithiothreitol, phenylmethylsulfonyl fluoride (PMSF), EDTA, EGTA, and leupeptin were obtained from Sigma. -Minimal essential medium (MEM), fetal bovine serum, trypsin-EDTA, and Geneticin were purchased from Life Technologies, Inc. Dowex AG 50 WX4 (200-400 mesh) and neutral chromatographic Alumina WN-3 were from Bio-Rad. DEAE-Sephacel was purchased from Pharmacia Biotech Inc. Carrier-free ²²NaCl (5 mCi/ml) and [-³²P]ATP (0.5 mCi/ml) were obtained from DuPont NEN. The different human N-terminal PTH and PTHRP analogues were kind gifts of Dr. K. Muller (CIBA-Geigy, Basel, Switzerland). Forskolin, 1,9-dideoxyforskolin, phorbol 12-myristate 13-acetate (PMA), 4-PMA, H-89, and chelerythrine chloride were purchased from Biomol Research Laboratories, Inc. (Plymouth Meeting, PA). All other chemicals were from Fisher and British Drug House Inc.

Chemically mutagenized Chinese hamster ovary (AP-1) cells devoid of endogenous Na⁺/H⁺ exchange activity (25) (kindly provided by Dr. S. Grinstein, Hospital for Sick Children, Toronto, Ontario) were maintained in MEM supplemented with 10% fetal calf serum, 100 µg/ml kanamycin sulfate, and 25 mM NaHCO₃, pH 7.4, and incubated in a humidified atmosphere of 95% air, 5% CO₂ at 37 °C.

The full-length rat PTH/PTHRP receptor type I cDNA (9) (generously provided by Dr. A.-B. Abou-Samra, Massachusetts General Hospital, Boston, MA) was subcloned into the mammalian expression vector pcDNA3 that contains the aminoglycoside phosphotransferase 3 gene which confers resistance to the antibiotic geneticin (G418 sulfate) (Invitrogen Corp., San Diego, CA). The rat Na⁺/H⁺ exchanger NHE3 isoform was subcloned into the vector pCMV as described previously (26). The cDNAs were transiently or stably transfected into AP-1 cells using the calcium phosphate-DNA coprecipitation technique of Chen and Okayama (27). AP-1 cells transiently expressing the PTH/PTHRP receptor type I were analyzed for ligand binding 72 h post-transfection. To select for stable expression of the PTH/PTHRP receptor type I, the cells were cultured in media containing G418 (600 µg/ml) starting 48 h after transfection and continued for a 1-2-week period. Surviving colonies were screened for their ability to activate adenylate cyclase activity in response to PTH-(1-34) stimulation. PTH-responsive AP-1 transfectants exhibiting the highest adenylate cyclase activity were subsequently transfected with the rat NHE3 cDNA. These cells were selected for Na⁺/H⁺ exchanger activity on the basis of their ability to survive an acute intracellular acid load as described previously (26). The transfected cells were maintained in standard culture medium supplemented with G418 (600 µg/ml).

Cells expressing the PTH/PTHRP receptor type I were measured for their ability to bind N-terminal peptide fragments of PTH and PTHRP by using a whole cell competitive binding assay (28, 29). Briefly, cells were plated at a density of 1 × 10⁵ cells/well in 24-well plates. Binding studies were carried out 24 h later following a 1-h incubation period in serum-free MEM. [¹²⁵I-Tyr³⁶]PTHRP-(1-36)-NH₂ was prepared by the lactoperoxidase technique and then purified by gel filtration chromatography. The cells were washed twice with 2 ml of Hanks' balanced salt solution before the addition of 0.2 ml of serum-free MEM medium containing 120,000 cpm of [¹²⁵I-Tyr³⁶]PTHRP-(1-36)-NH₂ per well. Unlabeled N-terminal peptide fragments of PTH and PTHRP (PTH-(1-34), PTH-(3-34), PTH-(28-42), PTH-(28-48), PTHRP-(1-34), and PTHRP-(1-16)) were added at varying concentrations (10¹⁰- 10⁵ M) in 0.3 ml of MEM to make a final incubation volume of 0.5 ml. Plates were incubated for 2 h at 37 °C. After incubation, cells were washed four times with Hanks' balanced salt solution before solubilization in 1 N NaOH and counting in a -radiation counter. Specific binding (B) was calculated as the difference between the total binding (<15% of total radioactivity) at each ligand concentration and the nonspecific binding (B_o) (~6% of total radioactivity) determined in the presence of excess nonradioactive, competitor ligand (10⁵ M). Values are presented as a percentage (P) of total specific binding (B_max). IC₅₀ values (concentration of competing ligand that resulted in 50% inhibition of the radioactive tracer ligand) were determined from plots of ln(P/(100  P)) (logit transformation of the sigmoidal binding data) as a function of the log(competitor ligand). The IC₅₀ is the concentration when logit = 0.

Adenylate cyclase activity was determined using a method based on incorporation of [³H]adenine into ATP and its conversion to [³H]cAMP as described previously (30). Briefly, confluent cells were incubated overnight in serum-free medium prior to each assay. The assay was initiated by adding [³H]adenine (4 × 10⁵ cpm) per cell culture well and incubating for 2 h. Following this incubation period, the medium was aspirated and the cells were washed twice with 1 ml of MEM without serum. Then, 0.5 ml of previously prepared solutions containing PTH or PTHRP analogues, forskolin, or PMA, in MEM supplemented with 1 mM isobutylmethylxanthine (to prevent breakdown of cAMP by phosphodiesterases) were added to each well, and cells were incubated for a period of 15 min. The medium of each well was then aspirated in the same order of application, and 0.5 ml of ice-cold 10% trichloroacetic acid was added to each well to stop the reaction and extract the [³H]cAMP. The [³H]cAMP was isolated by chromatography on Dowex and alumina columns and quantified in a -counter. Adenylate cyclase activity was expressed as [³H]cAMP produced per 15 min per well.

PKC activity was assayed according to previously described procedures (16, 31). Briefly, cells were washed twice with serum-free media and stimulated with different concentrations of N-terminal PTH or PTHRP analogues for a period of 2 min. At the end of the stimulation period, the cells were washed twice with ice-cold phosphate-buffered saline and then gently scraped in a buffer (800 ml) containing 2 mM Tris-Cl, pH 7.5, 250 mM sucrose, 2 mM EDTA, 5 mM EGTA, 1 mM DTT, 50 mM PMSF, and 2.5 mg/ml leupeptin. These cells were then sonicated twice on ice for a period of 10 s each using a Branson Sonifier (Model 450) set at low intensity. This was followed by a 60-min ultracentrifugation at 100,000 × g. The supernatant containing the cytosolic fraction was removed. The pellet containing the membrane fraction was resuspended in the same buffer (800 ml) containing 10% Triton X-100 and sonicated twice on ice for 10 s at medium intensity. The sonicate was shaken at 4 °C for 1 h and then subjected to ultracentrifugation at 100,000 × g for 60 min. The supernatant containing the solubilized membrane fraction was then collected. The cytosol and solubilized membrane fractions were then each applied to a DEAE-cellulose column that was washed with buffer containing 2 mM Tris-Cl, pH 7.5, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 50 mM PMSF, and 2.5 mg/ml leupeptin. The PKC enzyme was eluted from the column with buffers containing 130 and 150 mM NaCl. The PKC activity of the eluate was assayed by incorporation of [-³²P]ATP into a seven-amino acid synthetic peptide (FKKSFKL-NH₂) and quantified using a -counter. All counts were then corrected for the amount of protein present in 50 ml of the cytosolic or the solubilized membrane fraction. Results were calculated as the amount of PKC present in the membrane relative to the amount of PKC present in the cytosol, and control ratios were taken as basal activity and normalized to a value of 1.

Amiloride-inhibitable ²²Na⁺ influx was used as a measure of Na⁺/H⁺ exchanger activity as described previously (26). Briefly, transfected AP-1 cells were grown to confluence in 24-well plates. Cells were incubated in serum-free media overnight. Transfected cells were then incubated with isotonic NH₄Cl solution (25 mM NH₄Cl, 105 mM choline chloride, 5 mM KCl, 1 mM MgCl₂, 2 mM CaCl₂, and 20 mM HEPES-Tris, pH 7.4) for 30 min to acidify the cells to pH_i ~6.6 (pH_i was assessed by microfluorometry using the dye BCECF as described previously (32)). During the last 15 min of the NH⁺₄ prepulse, cells were treated with appropriate concentrations of N-terminal PTH or PTHRP analogues, forskolin, 1,9-dideoxyforskolin, PMA, or 4-PMA. The cells were then washed twice with a Na⁺-free choline chloride solution (130 mM choline chloride, 1 mM MgCl₂, 2 mM CaCl₂, 5 mM glucose, and 20 mM HEPES-Tris, pH 7.4). ²²Na⁺ influx assays were initiated by incubating the cells with the respective agents prepared in choline chloride solution containing 1 mM ouabain and 1 µCi of ²²NaCl (carrier free)/ml, and in the absence or presence of 1 mM amiloride for the indicated periods of time at room temperature. The nominal absence of K⁺ in the influx buffer and the presence of ouabain was used to prevent the transport of ²²Na⁺ catalyzed by the Na-K-2Cl cotransporter and Na,K-ATPase, respectively. The incubation was terminated (after 5 min of uptake) by adding 1 ml of ice-cold NaCl stop solution (130 mM NaCl, 1 mM MgCl₂, 2 mM CaCl₂, and 20 mM HEPES-Tris, pH 7.4). The solution was quickly aspirated and then rapidly washed an additional three times.

To extract the radiolabel, the cells were solubilized in 0.25 ml of 0.5 N NaOH and the wells washed with 0.25 ml of 0.5 N HCl. Both the solubilized cell extracts and the wash solutions were added to scintillation vials, and the radioactivity was measured in a -counter. Amiloride-sensitive Na⁺/H⁺ exchanger activity was defined as the difference between the rates of ²²Na⁺ influx in the absence and presence of 1 mM amiloride.

RESULTS

To directly assess the ability of the PTH/PTHRP receptor type I to bind N-terminal analogues of PTH and PTHRP (i.e. PTH-(1-34), PTH-(3-34), PTH-(28-42), PTH-(28-48), and PTHRP-(1-34)), Chinese hamster ovary AP-1 cells were transiently transfected with a full-length cDNA encoding the rat PTH/PTHRP receptor type I.

Cells transiently expressing the rat type I receptor specifically bound the radioligand [¹²⁵I-Tyr³⁶]PTHRP-(1-36)-NH₂ which was effectively competed off by increasing concentrations of nonradioactive PTH-(1-34) and PTHRP-(1-34) (IC₅₀  0.1-0.4 nM) (Fig. 1 and Table I). Similarly, PTH-(3-34), PTH-(28-42), and PTH-(28-48) produced a concentration-dependent reduction of [¹²⁵I-Tyr³⁶]PTHRP-(1-36)-NH₂ binding to the transfected cells, although they displayed a lower affinity for the receptor (IC₅₀  2-8 nM). In contrast, the N-terminal peptide analogue, PTHRP-(1-16), did not bind to this receptor, consistent with its lack of biological activity in renal and bone cell lines (7, 8). Untransfected AP-1 cells showed no specific binding to any of the PTH and PTHRP analogues (data not shown).

Table I. Competition binding of N-terminal PTH and PTHRP analogues to the rat PTH/PTHRP receptor type I expressed in AP-1 cells Analogue IC50 nm PTH-(1-34) 0.1  ± 0.01 PTHRP-(1-34) 0.4  ± 0.02 PTH-(3-34) 5.0  ± 0.3 PTH-(28-42) 2.0  ± 0.1 PTH-(28-48) 8.0  ± 0.8 PTHRP-(1-16) NDa a ND, binding not detected.

In order to further examine the ability of the PTH/PTHRP receptor type I to elicit biological responses, stably transfected cells were also examined. Unlike transiently transfected cells which must be analyzed within a narrow time frame, stable transfectants permit more long term studies to be conducted under more controlled, uniform conditions. Like acutely transfected cells, AP-1 cells stably expressing the type I receptor exhibited similar ligand binding kinetics using a subset of the analogues (i.e. PTHRP-(1-34), PTH-(3-34), and PTH-(28-48); IC₅₀ = 0.6, 5, and 1 nM, respectively). These cells were used for subsequent studies.

To assess the ability of the N-terminal PTH and PTHRP analogues to activate multiple signaling pathways, adenylate cyclase and PKC activities were assayed. As illustrated in Fig. 2A, PTH-(1-34) increased adenylate cyclase activity up to 7-fold in a concentration-dependent manner over the range of 0.01-100 nM, achieving half-maximal stimulation at approximately 0.1 nM. Based on this result, a single hormone concentration of 100 nM was selected to evaluate the effects of the other PTH and PTHRP analogues. As shown in Fig. 2B, 100 nM PTH-(1-34) and PTHRP-(1-34) stimulated adenylate cyclase activity 7-8-fold in AP-1 transfectants, whereas no increase in enzyme activity was detected with fragments PTH-(3-34), PTH-(28-42), PTH-(28-48), and PTHRP-(1-16), all lacking either the first two amino acids or amino acids 25-34. Forskolin (10 µM), a direct potent stimulator of adenylate cyclase, also increased adenylate cyclase activity to a similar extent (~12-fold), whereas the phorbol ester PMA had no effect. The latter result suggested that there is no cross-talk between PKC and the adenylate cyclase-PKA pathways, as has been observed in other cell types (33, 34).

In contrast to the effects on the adenylate cyclase system, significant hormone-stimulated translocation of PKC activity from cytosol to membrane was generally observed with all the N-terminal analogues at picomolar concentrations (i.e. 10 pM), except PTHRP-(1-16) which was inert in the PKC assay as it was in the adenylate cyclase assay (Fig. 3 A-C). As expected, translocation of PKC activity from cytosol to membrane was not observed in untransfected AP-1 cells, consistent with their lack of expression of PTH/PTHRP receptor activity (Fig. 3D). Interestingly, PTH-(1-34) was more effective in activating PKC at picomolar concentrations compared with its stimulation of adenylate cyclase. This is most likely explained by the differential sensitivities of the two signaling pathways to fractional occupancy of a single receptor. Therefore, the PTH/PTHRP receptor type I in transfected AP-1 cells is functionally coupled to both the PKA and PKC pathways.

Transduction of the second messenger signals induced by the N-terminal PTH and PTHRP analogues to downstream biological targets was assessed by measuring changes in the activity of the cotransfected Na⁺/H⁺ exchanger NHE3 isoform. This isoform has previously been found to be expressed in renal OK cells and inhibited by these PTH and PTHRP analogues via pathways involving PKA and/or PKC (7). In the present experiments, NHE3 activity was measured as initial rates of amiloride-inhibitable ²²Na⁺ influx following an acute, NH₄Cl-induced intracellular acid load (pH_i ~6.6). As illustrated in Fig. 4A, 100 nM concentrations of PTH-(1-34), PTHRP-(1-34), PTH-(3-34), PTH-(28-42), or PTH-(28-48) inhibited H⁺-activated NHE3 activity to levels of 63 ± 4, 65 ± 2, 86 ± 1, 80 ± 1, and 77 ± 1%, respectively, relative to control levels. The biologically inert analogue, PTHRP-(1-16), had no significant effect. These data implicated the involvement of PKC and possibly PKA in the pathway(s) leading to inhibition of NHE3. The inhibitory effects of these analogues were mimicked by 10 µM forskolin (76 ± 2% activity) and 1 µM PMA (72 ± 4% activity), whereas the corresponding biologically inert analogues, 1,9-dideoxyforskolin (10 µM) and 4-PMA (1 µM), had no effect.

Highly selective antagonists of PKC (i.e. chelerythrine chloride) (35) and PKA (i.e. H-89) (36) were used to confirm the signaling pathways involved in the inhibition of NHE3 activity. AP-1 transfectants were preincubated for 1 h with either 1 µM chelerythrine chloride or 100 µM H-89 followed by coincubation in the absence or presence of PTH analogues (100 nM), forskolin (10 µM), and PMA (1 µM). Chelerythrine chloride was ineffective in preventing the inhibition of NHE3 by PTH-(1-34), PTHRP-(1-34), and forskolin (Fig. 4A). However, it effectively abrogated the negative regulation elicited by PTH-(3-34), PTH-(28-42), PTH-(28-48), and PMA, agents that act exclusively through PKC. Likewise, H-89 alone had no influence on PTH-(1-34)- or PMA-mediated inhibition of H⁺-activated NHE3 activity, but it prevented the effects of forskolin (Fig. 4B). The latter result clearly indicated that H-89 was capable of selectively inhibiting the PKA pathway. H-89 also had no effect on the other N-terminal PTH analogues (data not shown). Treatment of these cells with 100 µM H-7, a protein kinase antagonist that inhibits PKA and PKC equally, prevented the depressive effects of PTH-(1-34) (Table II). These data indicate that the inhibitory action of PTH-(1-34) on NHE3 in these transfected cells involves both PKA and PKC.

Table II. Influence of the protein kinase inhibitor H-7 on PTH-(1-34)-mediated inhibition of Na+/H+ exchanger NHE3 isoform in transfected Chinese hamster ovary AP-1 cells Treatment NHE3 activity % Control 100  ± 3 PTH-(1-34) (100 nM) 64  ± 4a H-7 (100 µM) 96  ± 3 PTH-(1-34) + H-7 104  ± 4 a Significance from control values (p < 0.03).

DISCUSSION

The PTH/PTHRP receptor type I has been cloned from many tissues such as bone and kidney (9, 37, 38, 39) and belongs to a superfamily of G-protein-coupled receptors, including receptors for calcitonin, secretin, glucagon, glucagon-like peptide 1, growth hormone-releasing hormone, vasoactive intestinal peptide, gastric inhibitory peptide, corticotrophin-releasing factor A, and pituitary adenylate cyclase-activating peptide (40, 41, 42, 43). Common features of these receptors include similar membrane topology (i.e. seven membrane-spanning segments) and the ability to activate G-proteins that modulate the adenylate cyclase-cAMP-PKA and/or phospholipase C-diacylglycerol-PKC pathways.

Until recently, the ability of a single PTH/PTHRP receptor (i.e. type I) to couple to multiple effector systems was generally believed to account for the pleiotropic effects of PTH and PTHRP and their respective analogues in various target tissues (9). However, this paradigm is no longer tenable following the discovery of a second receptor, PTH2, that is expressed primarily in brain and pancreas and is distinguished by its ability to bind only PTH and to activate the PKA pathway (18).

Other lines of investigation also indicate that additional PTH/PTHRP receptors may exist. 1) N-terminal analogues of PTH and PTHRP activate PKC activity in ROS 17/2 osteosarcoma cells in a biphasic manner, with one peak of activity obtained at low picomolar concentrations and the other at nanomolar concentrations (16, 17, 44). Only the latter concentrations are coupled to adenylate cyclase activity. 2) Treatment of normal keratinocytes and squamous carcinoma cell lines with N-terminal peptide fragments of both PTH and PTHRP leads to increases in intracellular Ca²⁺ but not cAMP (19). The absence of a cAMP response is not a consequence of a dysfunctional signaling pathway, as squamous carcinoma cells stably transfected with the type I receptor show increased cAMP accumulation in response to PTH and PTHRP. These cells also express multiple mRNA transcripts that hybridize to type I receptor cDNA probes, yet differ significantly in size from the type I receptor mRNA present in human bone SaOS-2 cells. Differently sized transcripts are also observed in rat kidney, liver, skin, and testes (20). 3) C-terminal analogues of human PTH, such as PTH-(53-84), do not directly bind to the human PTH/PTHRP receptor type I and are unable to alter intracellular cAMP and Ca²⁺ levels, yet apparently retain the ability to stimulate alkaline phosphatase activity, osteoclast-like cell formation, and bone-resorbing activity by mature osteoclasts (21, 22, 23). 4) Likewise, C-terminal analogues of human PTHRP, such as PTHRP-(107-139), also do not bind to the PTH/PTHRP receptor type I, yet appear to signal by increasing intracellular Ca²⁺, but not cAMP, in hippocampal neurons (45). 5) Radioligand and affinity cross-linking studies have identified a 90-kDa protein in rat osteosarcoma (ROS 17/2.8) and rat parathyroid (PT-r3) cells that selectively binds with high affinity to the C-terminal region of PTH-(1-84) (46). 6) Apical and basolateral membranes isolated from rat renal cortical cells contain PTH/PTHRP receptors that differ quantitatively in their coupling to G-proteins and second messenger systems (47). These data have been interpreted to indicate the existence of a novel PTH/PTHRP receptor(s) or an alternatively spliced variant of the type I receptor, although other explanations, such as differences in the membrane environment or the signaling repertoire of the cell, may also explain some of the data.

Nevertheless, in view of the above observations, it was important to establish whether the regulation of the apical Na⁺/H⁺ exchanger NHE3 isoform by synthetic N-terminal analogues of PTH and PTHRP in renal OK cells could be solely accounted for by activation of the PTH/PTHRP receptor type I known to be expressed in this cell line. The data in this study clearly demonstrate that structurally diverse N-terminal analogues of PTH and PTHRP (i.e. PTH-(1-34), PTH-(3-34), PTH-(28-42), PTH-(28-48), and PTHRP-(1-34)) are able to directly bind to the rat PTH/PTHRP receptor type I, activate distinct second messenger systems, and elicit biological responses (i.e. inhibition of rat NHE3 activity) in a heterologous mammalian expression system.

Examination of these N-terminal analogues complement and extend previous studies that have tested the effects of PTH-(1-34), PTH-(3-34), or PTH-(7-34) on second messenger production in transiently transfected COS-7 cells (9, 37, 38) or stably transfected LLC-PK₁ cells (4) expressing either the opossum, rat, or human PTH/PTHRP receptor type I. In total, these data clearly establish that the N-terminal domains of PTH and PTHRP, despite having different amino acid sequences, probably share sufficient tertiary structure to bind to the type I receptor and activate the PKA and/or PKC pathways. Whether these analogues bind to the same or different regions of the receptor is unknown. The molecular mechanism by which some of these analogues can selectively activate PKC but not PKA is an area of particular interest. Recent structural studies of the type I receptor indicate that amino acids near the N terminus (residues 31-47) and within the third extracellular loop (residues 431-440) are important for ligand-receptor interactions (48, 49, 50), whereas the C-terminal cytoplasmic region between residues 480 and 591 influences G-proteins that regulate adenylate cyclase but not phospholipase C (51, 52).

In this study, significant differences were observed in the concentration of PTH-(1-34) required to induce PTH/PTHRP receptor type I activation of adenylate cyclase (K_0.5 ~10¹⁰ M) and PKC (K_0.5 <10¹¹ M) activities. As only one type of receptor is present in these cells, this difference appears to be an intrinsic feature of the protein and likely reflects differential sensitivities of the two signaling pathways to fractional occupancy of a single receptor. These kinetic differences mimic that observed in the opossum renal proximal tubule OK cell line which expresses endogenous PTH/PTHRP receptor type I and apical NHE3 activity (7, 37, 53).

Finally, our data indicate that the specialized apical membrane environment of OK cells is not a determining factor in the coupling of ligand-activated PTH/PTHRP receptor type I to inhibition of NHE3 activity. Although the results do not exclude the presence of other PTH/PTHRP receptors in OK and other renal proximal tubule cells, it suggests that the PTH/PTHRP receptor type I is sufficient to mediate the diverse biological actions of these N-terminal PTH and PTHRP analogues in these and possibly other cell types.