©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Signal Transduction by the Formyl Peptide Receptor
STUDIES USING CHIMERIC RECEPTORS AND SITE-DIRECTED MUTAGENESIS DEFINE A NOVEL DOMAIN FOR INTERACTION WITH G-PROTEINS (*)

(Received for publication, August 10, 1995; and in revised form, October 2, 1995)

Thomas T. Amatruda III (1)(§) Sasa Dragas-Graonic (1) Richard Holmes (2) H. Daniel Perez (2)

From the  (1)Division of Medical Oncology, Department of Medicine, University of Minnesota, Minneapolis, Minnesota 55455 and the (2)Department of Immunology, Berlex Biosciences, Richmond, California 94804

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The binding of small peptide ligands to high affinity chemoattractant receptors on the surface of neutrophils and monocytes leads to activation of heterotrimeric G-proteins, stimulation of phosphatidylinositol-phospholipase C (PI-PLC), and subsequently to the inflammatory response. It was recently shown (Amatruda, T. T., Gerard, N. P., Gerard, C., and Simon, M. I.(1993) J. Biol. Chem. 268, 10139-10144) that the receptor for the chemoattractant peptide C5a specifically interacts with Galpha, a G-protein alpha subunit of the G(q) class, to trigger ligand-dependent stimulation of PI-PLC in transfected cells. In order to further characterize this chemoattractant peptide signal transduction pathway, we transfected cDNAs encoding the formylmethionylleucylphenylalanine receptor (fMLPR) into COS cells and measured the production of inositol phosphates. Ligand-dependent activation of PI-PLC was seen in COS cells transfected with the fMLPR and Galpha and stimulated with fMLP but not in cells transfected with receptor alone or with receptor plus Galpha(q). Chimeric receptors in which the N-terminal extracellular domain, the second intracellular domain, or the intracellular C-terminal tail of the fMLP receptor was replaced with C5a receptor domains (Perez, H. D., Holmes, R., Vilander, L. R., Adams, R. R., Manzana, W., Jolley, D., and Andrews, W. H.(1993) J. Biol. Chem. 268, 2292-2295) were capable of ligand-dependent activation of PI-PLC when co-transfected with Galpha. A chimeric receptor exchanging the first intracellular domain of the fMLPR was constitutively activated, stimulating PI-PLC in the absence of ligand. Constitutive activation of PI-PLC, to a level 233% of that seen in cells transfected with wild-type fMLP receptors, was dependent on Galpha. Site-directed mutagenesis of the first intracellular domain of the fMLPR (amino acids 54-62) reveals this to be a domain necessary for ligand-dependent activation of Galpha. These results suggest that different receptors which mediate similar biochemical responses may utilize distinct mechanisms to activate G-proteins. Differences among the signaling pathways triggered by chemoattractant factor receptors suggest an opportunity for pharmacologic modifications of the inflammatory response.


INTRODUCTION

The responses of neutrophils and monocytes to bacterial infection are regulated by chemoattractant or proinflammatory factors, small peptide or lipid molecules that are ligands for high affinity receptors on the surface of inflammatory cells(1) . G-proteins, or heterotrimeric GTP-binding regulatory proteins, have been implicated in signal transduction of the response to chemoattractant ligands through biochemical studies of the effects of GTP analogues on chemoattractant factor binding and activation of phospholipases in permeabilized cells and membranes(2) . The ability of pertussis toxin (Bordetella pertussis islet-activating protein) to inhibit signaling by many chemoattractant ligands in neutrophils and monocytes implies a major role for pertussis toxin-sensitive G-protein heterotrimers in chemoattractant factor signaling(1, 3) . Two G-protein alpha subunit isotypes that are modified by pertussis toxin, G(i)alpha(2) and G(i)alpha(3), are expressed in inflammatory cells and have been proposed to regulate the pertussis toxin-inhibitable activation of PI-PLC (^1)observed in neutrophils and monocytes(1, 4) .

Chemoattractant peptides and other G-protein-coupled ligands also activate PI-PLC through pertussis toxin-resistant signal transduction pathways in inflammatory cells(5, 6) . We and others recently determined that Galpha, a G(q) class G-protein that is expressed in hematopoietic cells, specifically and potently interacts with the receptors for the chemoattractant peptide C5a to trigger ligand-dependent activation of PI-PLC in transfected cells (7, 8) . In the studies presented here we further explore signal transduction by this pathway. First, we examined the activation of PI-PLC by the receptor for the chemoattractant peptide fMLP in transfected cells. Then, we examined the role of specific domains of the fMLP receptor in signaling through the use of chimeric and mutagenized chemoattractant factor receptors.


MATERIALS AND METHODS

Transient Transfection and Labeling of COS-7 Cells

COS-7 cells (ATCC) were maintained, transfected, and labeled as described (7) , except that transfections were performed with Lipofectin or Lipofectamine (Life Technologies, Inc.). fMLP peptide (Sigma) was dissolved in dimethylformamide at a concentration of 1 mM, stored at -20 °C, and then added to inositol-free Dulbecco's modified Eagle's medium just before use. Cells were stimulated by incubation with fMLP peptide for 20 min, and then total inositol phosphates (IP) were measured as described(7, 9) . Mean levels of IP and standard error were calculated from assays on three separately transfected wells. The total amount of DNA transfected into cells was held constant by titration with a control plasmid. In co-transfection experiments, cells were transfected with expression plasmids consisting of receptor or G-protein cDNAs driven by the cytomegalovirus promoter at a molar ratio of 2:1 (receptor cDNA:G-protein cDNA). Under these conditions, Galpha is apparently present in excess, because the amount of the Galpha cDNA transfected into cells can be reduced by 50% without decreasing the ligand-dependent activation of PI-PLC (7) . (^2)Chimeric receptor was previously found to be expressed at 40-100% of the level of wild-type receptors(10) . In the analysis of data from independent experiments, IP production was normalized relative to total incorporation of [^3H]inositol into membrane phospholipids. Statistical comparisons were made using Student's t test (two-tailed).

Construction of COS Cell Expression Vectors

The human Galpha cDNA, murine Galpha(q), and bacterial beta-galactosidase expression vectors were as described(7, 9) . The human fMLP receptor clone used in initial studies was generously provided by C. Gerard. Chimeric fMLP receptor cDNAs were previously described(10) . Cassette alanine replacements within the first intracellular domain of the fMLP receptor were performed by oligonucleotide site-directed mutagenesis using a modification of the Altered Sites mutagenesis system (Promega)(10) . Equilibrium binding of iodinated fMLP to COS cells transfected with mutagenized fMLP receptors was performed at 4 °C as described (10) .


RESULTS AND DISCUSSION

Heterologous cells transfected with the receptor for the chemoattractant ligand fMLP have ligand binding properties similar to those seen in inflammatory cells(10, 11) . Although rapid activation of PI-PLC is seen in inflammatory cells after exposure to this ligand, COS cells expressing the fMLP receptor show only a slight ligand-dependent activation of PI-PLC (31 ± 9% increase in IP production (S.E., n = 8 experiments) (Fig. 1). In cells transfected with Galpha or with the fMLP receptor and Galpha, a 2-fold increase in base-line IP release was seen. Subsequent addition of fMLP ligand resulted in a dose-dependent increase in IP production, up to a maximal level of 349 ± 25%, the level seen in unstimulated cells (S.E., n = 17) (Fig. 1). As in the case of the C5a receptor(7) , activation of PI-PLC by the fMLP receptor was dependent on expression of Galpha, and no ligand-dependent activation of PI-PLC was seen after co-transfection of Galpha(q) with the fMLP receptor (Table 1).


Figure 1: Accumulation of [^3H]inositol phosphates in COS-7 cells transfected with the human fMLP receptor. COS-7 cells were transfected with the fMLP receptor or with the fMLP receptor plus Galpha, labeled, washed, and stimulated with medium alone (0) or fMLP ligand as described under ``Materials and Methods.'' Levels of IP were determined as described. Each shaded bar represents the mean of three assays from a representative experiment. Clear bars represent standard error.





These findings support previous reports that the signal transduction pathways triggered by fMLP and C5a are similar(12, 13) . In order to investigate the role of specific domains of these receptors in the activation of PI-PLC via the Galpha pathway, we transfected COS cells with cDNA clones, which encode chimeric chemoattractant factor receptors. The cell surface expression and ligand binding properties of these receptors have been previously described(10) . Fig. 2shows schematic diagrams of the receptor chimeras. The chimeric receptor in which the extracellular N-terminal domain of the fMLP receptor was replaced by the corresponding region of the C5a receptor (Ch 15) was capable of interacting with Galpha to stimulate hydrolysis of phosphatidylinositol. Addition of ligand resulted in an increase in IP production to a level 3.5 times that seen in unstimulated cells (Fig. 2). A similar pattern was seen in chimeric receptors in which the second intracellular loop (Ch 18) or the C-terminal tail (Ch 7) of the fMLP receptor were replaced with corresponding domains of the C5a receptor (Fig. 2). In cells transfected with the chimeric receptors, fMLP ligand induced activation of PI-PLC in a dose-dependent pattern but with an increased ED when compared with wild-type fMLP receptors expressed in COS cells (Fig. 3). Ligand-dependent stimulation of IP production by these receptors was dependent on co-expression of Galpha. As in the wild-type fMLP receptor, only a minor activation of PI-PLC was seen in COS cells transfected with chimeric receptors alone, and no ligand-dependent activation of PI-PLC was seen in cells transfected with receptors and with another G-protein of the G(q) class (Table 1).


Figure 2: Accumulation of inositol phosphates in COS-7 cells transfected with chimeric chemoattractant peptide receptors. COS-7 cells were transfected with a control beta-galactosidase plasmid (Lac) or with the Galpha cDNA and the following receptor clones: an fMLP receptor construct (10) or the chimeric receptors Ch 7, Ch 15, Ch 18, or Ch 16. Schematic diagrams of receptor clones show intracellular domains below and extracellular domains above. C5a receptor domains are marked in black. Cells were labeled and then stimulated with medium alone (0) or with 500 nM fMLP (+), and levels of inositol phosphate were measured. The figure shows results from a representative experiment. Each shaded bar represents the mean of three assays ± S.E. (clear bars).




Figure 3: Dose-response analysis of activation of PI-PLC by fMLP receptor chimeras. COS 7 cells were transfected with the Galpha cDNA and the fMLP receptor or the chimeric receptors Ch 7, Ch 15, or Ch 18. Cells were labeled and stimulated with fMLP ligand (0-500 nM), and then levels of IP production were determined. The figure displays IP production for each receptor clone relative to the maximal production seen when cells transfected with that clone were stimulated with 500 nM fMLP. Maximal IP production averaged 5,192 cpm. The figure represents results from three independent experiments ± S.E. of the mean, normalized relative to total incorporation of [^3H]myoinositol into membranes.



A contrasting pattern was seen in cells transfected with a chimeric fMLP receptor in which the first intracellular domain was replaced with the corresponding domain of the C5a receptor. In this receptor, Ch 16, the region of the wild-type fMLP receptor cDNA encoding the sequence LVIWVAGFRMTHTVTTISYLNKAVA was replaced with a portion of the C5a receptor cDNA which encoded LVVWVTAFEAKRTINAIWFLNLAVA. This chimeric receptor triggered activation of PI-PLC in the absence of ligand (Fig. 2). The level of IP production in cells transfected with this receptor and with Galpha was 233 ± 18% of the level seen in cells transfected with native fMLP receptors and Galpha (S.E., n = 14; p < 0.0001). Addition of fMLP ligand (5-500 nM) to cells expressing this receptor resulted in a further increase in IP production. The constitutive activation of PI-PLC by this receptor required co-expression of Galpha (Table 1).

The studies with chimeric receptor Ch 16 suggest that the first intracellular domain of the chemoattractant receptors plays a role in the activation of Galpha. Hydrophobicity plots predict that the residues RMTHTVTTI comprise the first intracellular domain of the fMLP receptor. In order to analyze the role of this region of the receptor in the activation of Galpha, we studied the signal transduction properties of fMLP receptors that were mutagenized by cassette alanine mutagenesis(20) . The activation of PI-PLC in cells transfected with Galpha and with the mutagenized receptors is shown in Fig. 4. Mutant receptor clone p301, encoding the sequence, RMTAAATTI, allowed ligand-dependent activation of PI-PLC at ligand concentrations of 50-500 nM. In contrast, the receptor p300 (AAAHTVTTI) triggered an attenuated increase in PI-PLC activation when stimulated with fMLP, and receptor p312 (RMTHTVAAA) did not activate PI-PLC when stimulated with ligand. None of the mutagenized receptors triggered constitutive activation of PI-PLC. These receptors had diminished ligand binding capacity; receptor p301 bound fMLP with a calculated K(d) of 50 nM, while receptors p300 and p312 bound ligand with K(d) of >50 nM, and equilibrium binding could not be demonstrated (data not shown).


Figure 4: Accumulation of inositol phosphates in COS-7 cells transfected with fMLP receptor mutants. COS cells were transfected with cDNA clones encoding beta-galactosidase (Lac), Galpha, fMLP receptor + Galpha, mutant receptors p300 + Galpha, p301 + Galpha, or p312 + Galpha. Cells were then labeled with [^3H]myoinositol and stimulated with fMLP peptide, 500 nM (+), or medium(-), and inositol phosphate production was measured as described. Each shaded bar represents the mean of three assays from a representative experiment. Clear bars represent standard error.



Chemoattractant peptide ligands can activate a G(q) class G-protein, Galpha, in transfected cells. The signal transduction properties of fMLP receptors reported here are similar to those described for the C5a receptor expressed in COS cells (7, 8) . The physiological role of Galpha in the response of inflammatory cells to these ligands has not been defined. While inhibition of the chemoattractant factor responses of inflammatory cells by PTX implies that the major signal transduction pathway activated by chemoattractant ligands is through G-protein heterotrimers including G(i)alpha(2) or G(i)alpha(3)(4, 13) , Galpha could account for aspects of the chemoattractant response which are not inhibited by PTX(1, 5, 6, 7, 12) .

Studies with chimeric chemoattractant peptide receptors may allow delineation of the functional roles of receptor domains in ligand binding and signal transduction(10, 14, 15) . Exchange of the N-terminal extracellular domain (Ch 15), second intracellular loop (Ch 18), or C-terminal tail (Ch 7) from the C5a to the fMLP receptor results in receptors that are capable of signal transduction via Galpha. These domains are proposed to be similar in overall topology and to share regions of primary sequence homology (13) . The chimeric receptors were previously shown to be expressed on the cell surface and to bind fMLP ligand. We noted an increase in the EC of these chimeric receptors, compared with wild-type fMLP receptors. This could reflect decreased ligand binding affinity or cell surface expression of the chimeric receptors (10) or may indicate alterations in the interactions of chimeric receptors with Galpha.

Our results suggest that the second intracellular domains of the fMLP and C5a receptors confer similar functional properties to these receptors. In earlier studies, replacement of the second intracellular domain of the fMLP receptor with the corresponding region of a receptor of similar structure, the FPR2 receptor, allowed ligand binding and calcium mobilization through a PTX-sensitive signaling pathway(14) . Synthetic peptides corresponding to this region of the fMLP receptor were found to interact with G(i)alpha(2) and possibly to inhibit the association of the fMLP receptor with G-protein heterotrimers(16, 17) . Together with the present findings, these results support the hypothesis that the second intracellular domain of chemoattractant peptide receptors contains a structural motif which is generally necessary for activation of G-proteins by seven-transmembrane domain receptors. A hydrophobic residue present in this domain both in the fMLP receptor (Leu-128) and the C5a receptor (Phe-139) has been proposed to be essential for activation of Galpha(q) by muscarinic and adrenergic receptors(18) .

Studies with chimeric receptor Ch 7 indicate that the C-terminal tail of the C5aR can substitute for the homologous region of the fMLPR in the activation of Galpha. Note that a region of the tail of the fMLPR (residues 323-339, LTEDSbulletTQTSDTATNSTL) is conserved in the C5aR (LTEESVVRESKSFTRSTV) (13) . This chimeric receptor was previously shown to bind fMLP ligand with high affinity. When expressed with Galpha, this receptor is capable of activation of PI-PLC in a ligand-dependent pattern and is not constitutively activated. Conservation of ligand binding and ligand-induced calcium mobilization has previously been demonstrated in chimeric receptors in which the C-terminal domain of the fMLP receptor was replaced with the analogous region of the FPR2 receptor(14) . In addition, a functional role for this domain of the fMLP receptor has been proposed from studies using synthetic peptides, which interfere with the interaction of receptors with Galphai(2) or Galphai(3)(16, 17) .

Our studies suggest that the first intracellular domain of chemoattractant peptide receptors regulates the activation of PI-PLC via the Galpha pathway. The constitutive activation of Galpha by chimeric receptor Ch 16 implies that the first intracellular domains of the C5a and fMLP receptors are not functionally equivalent, despite similar topology and some conservation of primary sequence. Previous studies of the first intracellular domain of the fMLP receptor have not indicated that it is involved in signal transduction. A receptor chimera in which the first intracellular domain of the fMLP receptor was replaced with a highly conserved homologous region of the FPR2 receptor showed a decrease in ligand binding affinity similar to that seen with fMLPbulletC5a receptor chimera Ch 16(14) . However, the fMLPbulletFPR2 receptor chimera did not trigger constitutive activation of calcium mobilization. Possible mechanisms to account for constitutive activation of the chimeric fMLPbulletC5a receptor Ch 16 include: (a) the first intracellular loop of fMLP receptor may function as an inhibitory domain, which constrains activation of the unliganded wild-type receptor(19) ; (b) the first intracellular loop of the C5a receptor may directly activate Galpha when removed from the context of the wild-type receptor; or (c) exchange of the first intracellular loop could disrupt the conformation of the receptor and indirectly unmask a receptor domain, which activates Galpha.

Mutagenesis of the first intracellular domain of the fMLP receptor confirms the involvement of this region of the receptor in signal transduction through Galpha. Each of these mutant receptors manifested dramatic decreases in ligand binding, suggesting that alterations in the first intracellular domain disrupted either the conformation of contiguous ligand-binding regions of the receptor or the receptor-G-protein interaction, which is necessary for high affinity binding. Previously, small changes in sequence in the first and second extracellular domains of the fMLP receptor were shown to disrupt ligand binding(10, 20) . Replacement of residues 54-56 and 60-62 with alanines attenuated or abolished the signaling properties of the receptors. These amino acids are predicted to sit at the intracellular face of the receptor, a region that has been implicated in G-protein activation through mutagenesis of several classes of receptors(21) . The observation that replacement of these residues did not lead to constitutive activation argues that the first intracellular domain of the fMLP receptor is not a specific inhibitory region that constrains receptor activation(19) . Our results are most consistent with the hypothesis that the first intracellular domain of both the fMLP and C5a receptors activates Galpha and predict that mutagenesis of the homologous region of the C5a receptor or the Ch 16 chimera will also decrease activation of Galpha by these receptors.

Our findings have several implications for the model of chemoattractant peptide signaling. First, we confirm that chemoattractant peptide ligands can activate PI-PLC through a G(q) class signal transduction pathway (Galpha). The physiological importance of this pathway remains undetermined. Second, our results confirm that exchange of certain domains of the fMLP and C5a receptor results in receptors that are capable of binding ligand and activating a G-protein signal transduction pathway. Third, we have demonstrated that the first intracellular domains of the fMLP and C5a receptors are not interchangeable and that the first intracellular domain of the fMLP receptor is necessary for activation of Galpha. The mechanism by which the chimeric receptor is constitutively activated remains unclear but apparently does not entail the elimination of a constraining or inhibitory region in the first intracellular domain of the fMLPR. The constitutive activation of Galpha by this chimeric receptor implies that these two receptors with apparently similar physiological actions differ in the mechanism by which they activate a G-protein pathway. This hypothesis receives indirect support from phylogenetic analysis. The first intracellular domain of the fMLP receptor is strongly conserved between mouse (FRMKHTVTT) and human (FRMTHTVTT)(13) , and the human fMLP receptor is capable of activating the murine homologue of Galpha, Galpha15.^2 In contrast, the primary sequence of the C5a receptor homologue from mouse (FEPDGPSNA) diverges from the human receptor in this region (FEAKRTINA)(13) . Understanding the physiological significance of these differences in receptor properties will require further insight into the role of Galpha in the physiology of cells of hematopoietic lineage.


FOOTNOTES

*
This work was supported in part by grants from the University of Minnesota, an institutional research grant from the American Cancer Society, and by a grant from the Leukemia Task Force, University of Minnesota. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Division of Medical Oncology, UMHC Box 286, University of Minnesota, Minneapolis, MN 55455. Tel.: 612-624-9127; Fax: 612-625-8966.

(^1)
The abbreviations used are: PI-PLC, phosphatidylinositol-phospholipase C; fMLP, formylmethionylleucylphenylalanine; fMLPR, formylmethionylleucylphenylalanine receptor; IP, inositol phosphate(s); PTX, pertussis toxin.

(^2)
T. T. Amatruda III and S. Dragas-Graonic, unpublished observations.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.