Introduction

Top Abstract Introduction Methods Results Discussion References

The histamine H2 receptor is important in the regulation of multiple physiological events extending from gastric acid secretion to tissue inflammation (Hill, 1990; Del Valle and Gantz, 1997). We and others have previously demonstrated that this G-protein coupled receptor can activate both the adenylate cyclase (Batzri and Gardner, 1978; Chew et al., 1980) and the phosphoinositide signaling pathways (Chew, 1986; Del Valle et al., 1992; Mitsuhashi et al., 1989). More recently, we have shown that H2 receptor coupling to these signaling pathways occurs via separate GTP-dependent mechanisms (Del Valle et al., 1992; Wang et al., 1996). Through site-directed mutagenesis and construction of chimeric proteins the putative intracellular domains involved in G-protein coupling and activation have been mapped for multiple receptors (Bonner, 1992; Fraser et al., 1994; Strader et al., 1994). The third intracellular loop (3iC) has been implicated as one of the principal determinants of receptor mediated G-protein activation (Eason and Liggett, 1995; O'Dowd et al., 1988; Strader et al., 1987; Wess et al., 1990). However, more recent studies suggest that several receptor segments besides 3iC play an important role in G-protein coupling. For example, mutation of the aspartic acid residue in the segment of the second intracellular loop (2iC) adjacent to 3iC results in a receptor with high ligand binding affinity but absent or reduced G-protein coupling (Eason and Liggett, 1995; O'Dowd et al., 1988). Similarly, multiple studies using site-directed mutagenesis and chimeric receptor techniques suggest that several components besides the 3iC are important in coupling to G-proteins that activate phospholipase C (Blin et al., 1995; Cotecchi et al., 1990; Hayashida et al., 1996; Nussenzveig et al., 1994; Wu et al., 1995).

Studies using synthetic peptides that correspond to different intracytoplasmic domains of several G-protein linked receptors (AR, 2A adrenergic, rhodopsin, N-formyl peptide, dopamine D2) confirm that multiple intracellular segments are important for G-protein recognition and activation (Bonner, 1992; Dalman and Neubig, 1991; Münch et al., 1991; Palm et al., 1990; Schreiber, 1994). It has been theorized that interaction between these loops is important for G-protein coupling (Cheung et al., 1992; Probst et al., 1992). Portions of the carboxyl terminus and third intracytoplasmic loop adjacent to the transmembrane domains of the receptor are thought to form amphiphilic -helices and facilitate G-protein coupling. Studies showing that the peptide mastoparan can activate G-proteins by forming an amphiphilic -helix at the inner surface of cytoplasmic membrane supports this theory (Hayashida et al., 1996; Higashijima et al., 1988).

Although a single H2 receptor can activate separate signaling pathways, the structural determinants required for G-protein coupling are unknown. To explore this question further we utilized a series of synthetic peptides corresponding to intracytoplasmic domains of the H2 receptor in an effort to map the regions important in G-protein activation.

	Methods

Top Abstract Introduction Methods Results Discussion References

Chemicals. Trichloroacetic acid, Triton X-100, forskolin, histamine, cimetidine, epinephrine, BSA, DTT, EDTA, IBMX were purchased from Sigma Chemical Co. (St. Louis, MO). EBSS was purchased from Irvine Scientific (Santa Ana, CA). Phosphatidylinositol 4,5-bisphosphate [(inositol-2-³H(N))PIP₂]([³H]PIP_2; 8.8 Ci/mmol₎ was purchased from Du Pont-New England Nuclear (Boston, MA). cAMP assay kits were from Amersham (Arlington Heights, IL). [methyl-3_H]tiotidine (87 Ci/mmol) was a product of Du Pont.

Peptide synthesis. Oligopeptides corresponding to the amino or carboxyl terminal regions of the second and third intracytoplasmic loops and the amino terminal segment of the carboxyl terminal tail of the canine H2 receptor (Gantz et al., 1991) were obtained from the University of Michigan Protein and Carbohydrate Structure Facility. A peptide corresponding to the amino terminal region of the receptor was used as a control (H2PC). Peptides were synthesized by a solid-phase Merrifield method on an automatic peptide synthesizer, then purified to >95% homogeneity by gel filtration and reverse-phase HPLC. The sequence of the synthetic peptides and the region of the receptor they correspond to are outlined in figure 1. 

View larger version (26K): [in this window] [in a new window]   Fig. 1.   Amino acid sequence of H2 receptor-specific peptides. Peptides encoding the identical sequence of the outlined regions of the canine histamine H2 receptor were synthesized H2PC was used as a control peptide

H2 receptor expression. The full-length coding region of the canine H2 receptor gene was subcloned into a CMVneo expression vector as previously described (Del Valle et al., 1992; Wang et al., 1996). HEPA cells (derived from a rat hepatoma) were transfected using the technique of calcium phosphate coprecipitation, and permanently transfected cells were selected by resistance to the neomycin analogue G418 (500 µg/l). Single clones of transfected cells were selected and screened for expression of the canine H2 histamine receptor by Northern blot analysis and receptor binding studies using [methyl-³H]tiotidine as the radioligand.

Membrane preparation. Membranes were prepared according to previously described methods (Wang et al., 1996). In brief, HEPA cells transfected with the histamine H2 receptor were resuspended in ice-cold 50 mM Tris-HCl (pH 7.4), 2 mM EDTA and 2 mM DTT and sonicated three times for 5 sec each. The sonicate was then centrifuged at 500 × g for 5 min to discard nuclei and the supernatant centrifuged at 54,000 × g for 10 min at 4°C. The pellet was resuspended in 10 mM Tris-HCl (pH 7.4) and 1 mM DTT then stored at -70°C.

Receptor binding studies. Receptor binding studies were performed as previously outlined (Wang et al., 1996). Membranes from transfected cells were prepared as outlined above and incubated with [methyl-³H]-tiotidine in the presence or absence of the histamine H2 receptor antagonist cimetidine (10⁴ M) for 30 min at 37°C. Binding reactions were terminated by centrifugation (540,000 × g at 4°C for 5 min) and the pellet washed three times with ice-cold PBS. Nonspecific binding was determined by the amount of radiolabel bound in the presence of maximum concentrations of cimetidine (10⁴ M) and specific binding was calculated by subtracting nonspecific binding from maximum bound radioactivity.

Measurement of adenylate cyclase activity. Adenylate cyclase activity in membranes derived from transfected HEPA cells was measured using previously described methods (Wang et al., 1996). Membrane protein (80 µg) was added to an assay mixture (100 µl) containing 1 mM EDTA, 5 mM MgCl2, 0.5 mM ATP and 1 mM DDT and incubated at 37°C for 20 min. Ice-cold trichloroacetic acid (30%) was added to stop the reaction and precipitate cellular protein. The precipitate was centrifuged for 10 min at 1900 × g, and the supernatant was ether extracted, lyophilized and resuspended in 50 mM Tris-HCl (pH 7.4) and 2 mM EDTA. cAMP content was measured by competitive protein binding assay (Amersham).

Measurement of PLC activity. PLC activity in membranes was measured according to previously described methods (Wang et al., 1996). The assay mixture consisted of 30 µl of assay buffer containing 50 mM N-2-hydroxyethylpiperzine-N'-2-ethanesulfonic acid (pH 7.0), 100 mM KCl, 6 mM MgCl₂, 0.6 mM CaCl₂, and 2-ethylene glycol-bis(-aminoethy ether)-N',N',N',N'-tetra acetic acid], 20 µM [³H]-PIP2 [15,000 counts/min (cpm)], 30 to 50 µg of membranes and incubated at 37°C for 15 min. The reaction was terminated by adding 0.5 ml chloroform/methanol/HCl (100:100:0.6), followed by adding 0.15 ml 1 N HCl (+5 mM EGTA). Samples were vortexed and centrifuged, and radioactivity was quantified in 200 µl of the aqueous phase.

Measurement of peptide activity. The effect of synthetic oligopeptides was tested by incubating isolated membranes with each of the ligands for varying time intervals (10-120 min) and at varying temperatures (4 and 37° C). For stimulated studies, membranes were preincubated with peptides for 15 min before treatment with specific agents.

Statistical analysis. Data are presented as mean ± S.E., where n is equal to the number of cell preparations examined. Statistical analysis was performed using either Student's t test or analysis of variance if multiple comparison were performed and P < .05 was considered significant.

	Results

Top Abstract Introduction Methods Results Discussion References

Effect of H2 specific peptides on tiotidine binding and basal signal transduction. We first characterized the membrane preparation for receptor binding and secretogogue mediated signaling. [³H]-tiotidine bound to transfected HEPA cell membranes in a specific manner. Pretreatment of cell membranes for 30 min with individual peptides did not alter specific binding of [³H]-tiotidine. Similarly, combination of the five peptides (H2P2iN, H2P2iC, H2P3iN, H2P3iC and H2P4iN) did not alter specific binding or the IC50 for cimetidine (fig. 2). Basal AC and PLC activity in this preparation was 2.2 ± 0.15 pmol/min/mg protein (mean ± S.E., n = 6) and 125.5 ± 2.5 dpm/min/mg protein (mean ± S.E., n = 6). Histamine (10⁴ M) stimulated AC (10.4 ± 2.5 pmol/min/mg protein, n = 8), and PLC (250.4 ± 23.5 dpm/min/mg protein, n = 6) activity in transfected Hepa cell membranes. The synthetic peptides H2P2iN, H2P2iC, H2P3iN, H2P3iC, H2P4iN and H2PC did not alter basal signaling in our preparation despite treating membranes for up to 120 min and varying the incubation temperature (4 or 37°C) (fig. 3A and B).

View larger version (21K): [in this window] [in a new window]   Fig. 2.   Effect of H2-specific peptides on [3H]-tiotidine binding. Pretreatment of HEPA cell membranes with the combination of H2P2iN, H2P2iC, H2P3iN, H2P3iC and H2P4iN, each at a concentration of 104M, did not alter binding of [3H]-tiotidine. Data are expressed as mean ± S.E. of tiotidine binding (% B/Bo) where n = 4 separate experiments.

View larger version (24K): [in this window] [in a new window]   Fig. 3.   Effect of H2 specific peptides on basal adenylate cyclase (AC) and phospholipase C (PLC) activity. Each of the peptides was used at a concentration of 104 M. Peptides corresponding to 2i, 3i or the C-terminal tail failed to alter basal AC or PLC activity in HEPA cell membranes. Data are expressed as mean ± S.E. of six separate experiments.

Effect of synthetic peptides on histamine stimulated AC activity. H2P2iN, H2P2iC, H2P3iN and H2P4iN did not alter histamine-stimulated AC activity. Contrary to this, H2P3iC inhibited histamine's action to 69.7 ± 3.3% of maximal stimulation (mean ± S.E., n = 6) (fig. 4A). To further assess the specificity of our findings we examined whether the modest inhibitory effect of H2P3iC was dose dependent. As shown in figure 4B, the peptide corresponding to the carboxyl terminus of the third intracytoplasmic loop inhibited histamine stimulated AC activity with an IC50 of 3.5 × 10⁶ M. 

View larger version (31K): [in this window] [in a new window]   Fig. 4.   H2 receptor-specific peptide-mediated inhibition of histamine-stimulated AC activity. A, Each of the peptides was tested at a concentration of 104 M. H2P3iC inhibited histamine (105 M) stimulated AC activity to 69.7 ± 3.3% of maximal stimulation. B, The inhibitory effect of H2P3iC was dose dependent with an IC50 ~ 3.5 × 106 M. Data are expressed as the mean ± S.E. of % maximal stimulation where the asterisk represents P < .05 as compared to maximal stimulation.

5

View larger version (28K): [in this window] [in a new window]   Fig. 5.   Effect of combining H2 receptor-specific peptides on histamine-stimulated AC activity. Each peptide was used at a concentration of 104M. A, Combination of the peptides corresponding to the 2i and 3i nearly abolished histamine-mediated action. B, The inhibitory effect of the peptide combination was dose dependent. Data are expressed as the mean ± S.E. of % maximal stimulation where the asterisk represents P < .05 as compared to maximal stimulation.

View larger version (25K): [in this window] [in a new window]   Fig. 6.   Effect of H2-specific peptides (104 M for each peptide) on histamine (His, 104 M), GTPs (106 M), forskolin (Forsk, 105 M) and epinephrine (Epi, 105 M) stimulated AC activity. H2-specific peptides did not inhibit GTPs, forskolin or epinephrine stimulated AC activity. Data are expressed as the mean ± S.E. of AC activity (pmol/min/mg protein), where the asterisk represents P < .05 as compared to maximal activity.

Effect of synthetic peptides on histamine stimulated PLC activity. H2P2iN, H2P2iC, and H2P4iN had a slight, but significant, inhibitory effect on histamine-stimulated PLC activity although H2P3iN and H2P3iC inhibited histamine-mediated action to a greater degree (fig. 7A). The effect of these peptides was dose dependent (fig. 7B), with H2P3iC having a lower IC50 (7.5 × 10⁷ M) than H2P3iN (1.26 × 10⁵ M). The specificity of peptide-mediated action was further confirmed by demonstrating that they did not inhibit GTPs or AVP-stimulated PLC activity (fig. 8).

View larger version (27K): [in this window] [in a new window]   Fig. 7.   Effect of H2-specific peptides on histamine-stimulated PLC activity. A, Peptides (104 M) corresponding to 2i and 3i inhibited histamine stimulated PLC activity. Peptides corresponding to the C-terminus of the third intracytoplasmic loop inhibited PLC to the greatest degree. B, The inhibitory effect of both H2P3iN and H2P3iC was dose dependent with IC50s of 1.2 × 105 M and 7.5 × 107 M, respectively. Data are expressed as the mean ± S.E. of % maximal stimulation where the asterisk represents P < .05 as compared to maximal levels.

View larger version (32K): [in this window] [in a new window]   Fig. 8.   Effect of H2P3iC (104 M) on histamine (His, 105 M), vasopressin (AVP, 107 M) and GTPs (105 M) stimulated PLC activity. H2P3iC only inhibited histamine's effect on PLC activation. Data are expressed as the mean ± S.E. of PLC activity (dpm/min/mg protein) where the asterisk represents P < .05 as compared to histamine-stimulated levels.

	Discussion

Top Abstract Introduction Methods Results Discussion References

We have used synthetic oligopeptides corresponding to intracytoplasmic segments of the H2 receptor in an effort to characterize the structural requirements for histamine-mediated dual signaling. Our studies suggest that the second and third intracytoplasmic loops of this receptor are involved in activation of both the adenylate cyclase and phosphoinositide pathways. Moreover, it appears that distinct segments of these loops have differential effects on the two signaling pathways examined.

Insight into heptahelical receptor-G-protein coupling has been gained through the application of receptor based site directed mutagenesis and chimeric receptor construction. Numerous studies have demonstrated that the third and second intracytoplasmic loops, in addition to the carboxyl terminal tail, are important for regulating G-protein activity. It appears that the relative importance of each segment will vary according to the individual receptor. Mapping of receptor structure involved in regulating signaling cascades has also been performed through the use of receptor specific peptides. Studies by König and coworkers (1989) demonstrated that peptides corresponding to the second and third intracellular loops and the amino terminus of the carboxyl terminal tail blocked binding of the rhodopsin receptor to Gt. A number of control peptides corresponding to additional surface regions of this receptor failed to block binding to Gt. Synergism for inhibition was observed when the effective peptides were combined, suggesting the involvement of more than one receptor segment in G-protein coupling.

The synthetic peptides we examined did not stimulate basal AC or PLC activity. These results are at odds with several reports demonstrating that receptor specific synthetic peptides can activate specific G-proteins (Cheung et al., 1991; Münch et al., 1991; and Palm et al., 1990). Okamoto et al. (1991) hypothesized that peptides which activate G-proteins often have three characteristics including a length of 10 to 26 amino acid residues, two basic amino acids at the amino terminus and a BBXB or BBXXB (B = basic amino acid) sequence at the carboxyl terminus. As noted in figure 1, our H2-specific peptides did not fulfill all of these criteria. The lack of sequence conservation with other well-characterized receptor specific peptides may account for the lack of AC or PLC activation in our system.

Our observation that the 3iC-specific peptide inhibited histamine-stimulated AC activity suggests that this region is important for Gs activation, but the modest decrease noted supports the theory that other receptor segments may play an important role in histamine signaling. This assumption is confirmed by our results illustrating the synergism between peptides. Addition of the peptide corresponding to the amino terminus of 3i appeared to slightly enhance H2P3iC action, but statistical significance was not achieved. Significant enhancement of H2P3iC was achieved only after addition of synthetic peptides corresponding to 2iC. The combination of the four peptides essentially abolished histamines action suggesting that both loops are important in H2 receptor-mediated recognition and activation of Gs. Our findings demonstrating the importance of multiple intracytoplasmic sites of the H2 receptor for Gs activation are consistent with observations made with other G-protein coupled receptors (Hawes et al., 1994; König et al., 1989; and Münch et al., 1991).

Contrary to our AC studies, individual peptides corresponding to either of the segments tested had a significant inhibitory effect on histamine-stimulated PLC activation. Of these it appeared that peptides corresponding to the third intracytoplasmic loop (H2P3iN and H2P3iC) had the greatest inhibitory effect with H2P3iC blocking PLC activity to 20.8 ± 6.3% of maximal stimulation. The inhibitory effect of these two peptides on PLC activity was dose dependent with an IC50 of 1.2 × 10⁵ M and 7.5 × 10⁷ M for H2P3iN and H3P3iC, respectively. The greater potency and efficacy of H2P3iC over H2P3iN suggests that the carboxyl terminal tail of the third intracytoplasmic loop is most important for activation of the phosphoinositide pathway. Our findings are in part consistent with those observed with the angiotensin II type 1 receptor (AT2R1) (Wang et al., 1995) and the PAFR (Carlson et al., 1996), both of which couple to the phosphoinositide signaling pathway. Chimeric receptor studies by Wang and co-workers (1995) suggest that the third intracytoplasmic loop of AT2R1 is essential for activation of Gq. Contrary to our studies, it appears that the N terminus is more important than the C terminus of 3i. These investigators also observed that the intermediate portion of this loop was not important for signaling. Carlson and co-workers (1996) transfected individual minigene constructs containing each of three intracellular domains of PAFR and demonstrated that only the 3i expressing construct could inhibit PAF-stimulated IP production. These investigators also designed chimeric receptor experiments in which the 3i domain of PAFR was engineered into the corresponding segment of the rat pituitary adenylate cyclase-activating polypeptide receptor (which only activates adenylate cyclase) conveying the ability to activate PI turnover. Together, these studies suggest that 3i of PAFR is an important determinant of PI activation.

We used several parameters to assess the specificity of peptide-mediated inhibition of H2 receptor signaling. A control peptide corresponding to an extracellular domain of the H2 receptor did not alter histamine-mediated signaling. Our observation that peptides inhibited histamine-stimulated AC and PLC in a differential manner is indirect evidence supporting peptide specificity. Peptide concentrations with which we observed an inhibitory effect are consistent with previously published studies (Cheung et al., 1991; Dalman and Neubig, 1991; König et al., 1989; Münch et al., 1991; Palm et al., 1990). Finally, H2-specific peptides failed to alter epinephrine, GTPs and forskolin-stimulated AC activity or GTPs and vasopressin-stimulated PLC.

Our studies provide insight into the mechanism through which one receptor may couple to multiple G-proteins. As observed previously with other receptor models, H2 receptor activation of AC and PLC pathways involves the interaction of multiple receptor segments with corresponding G-proteins. We also observed differences in the requirements for activation of AC and PLC. Although 3iC appears to be involved in the action of both AC and PLC, it appears that the interaction of multiple receptor segments is not as important for stimulation of the latter. The near complete inhibition of PLC stimulation with 3i corresponding peptides suggests that this segment is most critical for phosphoinositide signaling. Studies with the dual coupling (PLC and AC) TSH receptor (Kosugi et al., 1993) indicate that the N and C terminal five amino acid residues of 3i are important for PLC activation but not cAMP regulation. Our results are somewhat consistent with these findings, although 3i also appears to play a role in AC stimulation. The specific amino acids required for differential coupling of the H2 receptor to separate pathways remains to be established. In conclusion, our data suggest that the ability of a single receptor to activate more than one signaling pathway may be dictated in part by differential coupling between different receptor segments and selected G-proteins.