Complement factors and their receptors

Immunopharmacology ELSEVIER

Immunopharmacology38 (1997) 3- 15

Review article

Complement factors and their receptors Julia A. Ember, Tony E. Hugli * Department of Immunology, The Scripps Research Institute, 10666 North Torrey Pines Road, La Jolla, CA 92037, USA

Received 15 August 1997

Keywords: Anaphylatoxin;C3a; C5a; C3a receptor; C5a receptor

1. Introduction The physiologic role of complement (C) components in immune and inflammatory responses continues to be a major field of study. Recent development of more sophisticated molecular and biochemical technologies has made it possible to explore and identify new biologic phenomena and confirm known functions associated with the various complement factors. It is an exciting time to observe discovery of the many novel functions and actions being attributed to components derived from this complex humoral system. The role of complement in clinical disease and host defense gains stature with each new biological finding. The 30 odd complement components that make up the system include proteolytic pro-enzymes, nonenzymatic components that form functional complexes, co-factors, regulators and receptors (see Fig. 1 and Table 1). It is the pro-enzymes which become sequentially activated in a cascade of events leading to activation of the complement system. An overview of the major components of the serum complement system and their activation pathways are presented in

* Corresponding author. Tel.: + 1-619-7848158; fax: + 1-6197848307.

Fig. 1. The proteolytic cascade allows for a tremendous amplification, since each proteinase molecule activated at one step can generate multiple copies of an activated enzyme later in the cascade, which in turn cleaves non-enzymatic components such as C3, C4 and C5. A number of the complement products generated during activation play a major role in host defense. The larger fragments derived from C3 and C4 (i.e. C3b and C4b) are involved in biologic effector functions, such as in opsonization, phagocytosis and immunomodulation. The smaller fragments, C3a, C4a and C5a, called anaphylatoxins, are involved in mediation of inflammatory reactions. The promotion of cellular activation and lytic events by a complex composed of the 'late' C5b to C9 components (i.e. membrane attack complex, MAC) are all well documented in the literature. Many of the mechanisms underlying these functions have also been described in detail thanks to the 'molecular revolution' of the past decade. There are several articles being presented in this Special Complement Issue that deal with biological or molecular aspects of various complement components and their receptors other than the anaphylatoxins. In addition, several recent and comprehensive reviews exist describing the C cascade and all of its intricate beauty (Reid, 1986; Rother and Rother, 1986; Frank, 1987; Kazatchkine

0162-3109/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S0162-3109(97)00088-X

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J.A. Ember, T.E. Hugli/lmmunopharmacology 38 (1997)3-15

Overview of Complement Activation Pathways Classical Pathway Antigen-Antibody (IgG or IgM) Complex C1

> ActivatedC1 (Ch~ssk~ Pathway C3 convertase)

(C~¢,sicalPathway CS convertase)

(Altemative Pathway Factor : C3 convertase) B Factor D

(AlternativePathway C5 convertase)

,~

Alternative Pathway

Fig. l. The Classical Pathway is initiated by C1 binding to antigen-antibody complexes or aggregated forms of the immunoglobulins. The Alternative Pathway is initiated by C3b binding to various activating surfaces including microbial cell walls and complex polysaccharides. The C3b involved in Alternatiue Pathway initiation may be generated in several ways, including spontaneous cleavage of a thioester by amino groups, hydroxyl groups and water. Both pathways converge at the C3 convertase step leading to C5 cleavage and self-assembly of the membrane attack complex (MAC). The MAC functions by penetrating a cell membrane and causing cellular lysis. Bars over the complexes designate enzymatic activity. The products shown in the boxes (C3a, C4a and C5a) are the anaphylatoxins.

Table 1 Complement cofactors, regulators and receptors Component

Function

Co-factors / regulators Properdin Factor H CIlNH Carboxypeptidase N C3b/C4b-binding proteins Factor I DAF MCP (gp 45,70) (CD46) Protein S

stabilizes the complex (C3b, Bb) co-factor for Factor I and binds to C3b to compete with Factor B binding inhibits active C 1s an anaphylatoxin inactivator that removes a C-terminal arginyl residue co-factor for Factor I and binds to C3b/C4b to compete with Factor B binding enzymatically cleaves C3b to C3bi accelerates decay of (C4b, C2a) and (C3b, Bb) co-factor for Factor I binds to C5b-C7 and prevents MAC formation

Receptors ClqR CRI (CD35) CR2 (CD21) CR3 (CD1 la and b) CR4 (P150,95, CD1 lc) C3aR C5aR(CD88)

enhances cell-mediated cytotoxicity and IgG-mediated phagocytosis binds C3b and has co-factor and decay accelerating activity binds to C3bi, C3dg and C3d fragments, has co-factor and immunomodulatory activity binds to C3bi and enhances phagocytosis recognizes the RGD sequence and binds C3bi mediates cell activation signals to C3a/C4a? mediates cell activation signals to C5a

J.A. Ember, T.E. Hugli / lmmunopharmacology 38 (1997) 3-15

and Carreno, 1988; Miiller-Eberhard, 1988; Frank, 1989; Lachmann, 1990; Farries and Atkinson, 1991; Kinoshita, 1991; Johnston, 1993; Moffitt and Frank, 1994; Barnum, 1995; Gardinali et al., 1995; Ward, 1996; Carroll and Fischer, 1997). Because this is such a vast and complicated field, we will discuss only the anaphylatoxins and anaphylatoxin receptors. An impressive list of functions assigned to complement products over the years have been associated with the smaller bioactive fragments released from C3, C4 and C5 during complement activation, fragments that are known as the anaphylatoxins. Anaphylatoxins play major a role in inflammation, including the recruitment and activation of various leukocytes. C3a is a 9 kDa peptide fragment released during selective proteolytic cleavage of the C3 a chain by a C3 convertase of the classical or the alternative pathway. C4a is a 8.7 kDa peptide released from the a chain of C4 by C2a cleavage as an early step in the classical pathway. C5a is a 11 kDa peptide released from the a chain of C5 by action of either classical or altemative pathway C5 convertases (see Fig. 1) There is renewed excitement and interest in the area of anaphylatoxin research because of recent advances in the characterization of anaphylatoxin receptors. Molecular data have recently provided information such as cloning of the C5a receptor (C5aR) (Boulay et al., 1991; Gerard and Gerard, 1991) and the C3a receptor (C3aR) (Roglic et al., 1996; Ames et al., 1996; Crass et al., 1996). For example, we learned that the C3aR has an unusually large extracellular loop, which represents an unique structural characteristic compared with most other G-protein coupled receptors, including the C5aR. Recent evidence that C5a plays an important role in immune injury in the lung (Mulligan et al., 1996, 1997; Schmid et al., 1997) and in post-ischemic vascular and tissue injury (lto and Del Balzo, 1994; Amsterdam et al., 1995; Ivey et al., 1995) supports the contention that regulation of selected complement activation products may be of therapeutic value. Both C3aR and C5aR exist on numerous cell types other than circulating white cells, such as hepatocytes, lung epithelial cells (Haviland et al., 1995), endothelial cells (Foreman et al., 1994), and even the astrocytes and microglial cells in brain tissue (Gasque et al., 1995, 1997). The wide-spread distribution of

5

anaphylatoxin receptors has serious implications for these factors playing a significant role in vascular, pulmonary and degenerative neurologic diseases. As we leam more about the impact of complement fragments on cellular and tissue functions, it will become apparent both when, and to what extent, these humoral factors integrate into normal host defense mechanisms versus how they may promote or exacerbate pathologic conditions.

2. Anaphylatoxins C3a, C4a and C5a Comparisons of primary structures of C3a, C4a and C5a has thought us that, although these molecules have common genetic ancestry, they have markedly different sequences. Only 13 residue positions have been totally conserved between C3a, C4a and C5a molecules from the various species presently analyzed, with 6 of these positions being the immutable cysteinyl residues that direct molecular folding. This level of sequence diversity is manifested by a failure of antibody to anaphylatoxin from one species to fully cross-react with the same anaphylatoxin from another species. The greatest homology in these structures exists adjacent to the two Cys-Cys sequences that occur at approximately the same location in each of these molecules. The relatively conserved C-terminal portion in each anaphylatoxin defines a unique effector site for these molecules. The C-terminal pentapeptide sequence - L - G - L - A - R in C3a is conserved for all known C3a molecules, - A / V - G / H - L - A / Q - R is a characteristic effector sequence for C4a, and -M/I/V-Q-L-G-R represent a relatively conserved effector sequence in C5a. Studies based on limited proteolytic degradation of the natural factors (Gerard et al., 1979; Chenoweth et al., 1980), and results obtained with bioactive synthetic analogues that mimics the sequences of these C-terminal regions, provided further evidence that the C-terminal portion of these molecules represent sites essential for mediating effector function. Thus the primary structural data for these molecules contributed a foundation for attempting structure/function analyses, as well as an opportunity for mapping ligand/receptor interactions. Recognition that one could synthesize fragments

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J.A. Ember, 72E. Hugli / lmmunopharmacology 38 (1997) 3-15

capable of exhibiting a full range of anaphylatoxin activities was first reported in 1977 (Hugli and Erickson, 1977). It was this report that provided clear evidence of bioacfivity associated with synthetic peptides based on the C-terminal sequence of the anaphylatoxins. In recent years, numerous synthetic homologues and analogues of C3a, C4a and C5a molecules have been designed and many have exhibited relatively high levels of potency. These peptides have generally been full agonists of the respective anaphylatoxin on which the analogue structure was based.

3. Synthetic analogues of anaphylatoxins Numerous advantages are realized in using synthetic analogues of the anaphylatoxins in place of the natural factors. In addition to confirming biologic responses obtained with the natural factors, large quantifies of synthetic peptides with high purity can be produced for investigative use. The peptides have greater overall stability than do the natural factors, and there is better control over contamination by other factors and endotoxins. Above all of these advantages, synthetic analogues of these effector molecules provide ideal tools for analyzing detailed ligand-receptor interactions. 3.1. C3a

Although no more than 5 to 8 residues of the C-terminal portion of C3a were required for biologic (spasmogenic) activity (Hugli and Erickson, 1977), potency is markedly enhanced as the length of the C-terminal C3a peptides is increased (Caporale et al., 1980). A synthetic, 21-residue C-terminal fragment of C3a (i.e. C3a 57-77) expressed approximately equipotent spasmogenic activity on guinea pig tissue to that of natural human C3a (Table 2a; Caporale et al., 1980; Lu et al., 1984). This suggested that interaction between the C-terminal 21 residues of C3a and the receptor binding site, is sufficient to elicit cellular activation. This 2 l-residue fragment of C3a was shown to assume a partial helical conformation in trifluoroethanol, a helix inducing solvent (Lu et al., 1984). Analogues of the 21-residue C3a peptide (C3a 57-77), containing various substituted

residues that disrupt helix formation exhibited less potency than C3a 57-77. Consequently incorporation of helix promoting residues in the 21-mer enhanced potency above that of C3a 57-77 (Hoeprich and Hugli, 1986). Synthetic C-terminal C3a peptides that were shorter than the tridecapeptide C3a 65-77 assumed irregular a n d / o r beta-tum-like conformations in trifluoroethanol, compared with the 21-residue analogue C3a 57-77 that is partially helical (Lu et al., 1984). More recently, NMR studies of human C3a suggested that the long C-terminal helix extends from residues 49 to 6 5 / 6 6 and that the remaining 11-12 residues at the C-terminal end assume an irregular conformation (Kalnik et al., 1991). These data suggest that the longer and more potent synthetic analogues of C3a may better mimic the conformation of the C-terminal region of the native molecule. In a more hydrophobic environment than water, such as at the surface of the membrane or on the C3a receptor, the longer (i.e. 21-residue) synthetic C3a peptides may readily assume helical structure like that in the natural factor, thus promoting their effectiveness. Substituted C3a analogue peptides have recently been designed that are considerably shorter than the 21-residue peptide, but are actually more potent in some biologic assay systems than C3a 57-77 (Gerardy-Schahn et al., 1988, 1989; Ambrosius et al., 1989; KiShl et al., 1990; Kola et al., 1992). This enhanced activity was achieved simply by attaching a hydrophobic fluorenyl-methoxycarbonyl (Fmoc) or 2-nitro-4-azidophenyl (Nap) group to the N-terminus of the 5- to 13-residue C-terminal C3a pepfides (Table 2a). Two- to ten-fold increases in biologic potency for the Fmoc- and Nap-modified C3a peptides, over that of C3a 57-77, were obtained using a guinea pig platelet ATP-release assay. A systematic study was conducted to determine an optimal distance for placing a hydrophobic group adjacent to the C-terminal effector site in the C3a peptide. Analogue C3a peptides of 10-18 residues in length were evaluated using guinea pig platelet aggregation and vascular permeability assays. The natural amino acid tryptophan was used as the N-terminal hydrophobic group in a series of peptides of 10-18 residues in length, and the 13-residue analogue exhibited the highest potency (Table 2b). The peptide WWGKKYRASKLGLAR was approximately 16 times more potent than the 21-re-

(native sequence)

100 120 325 604 1271 1406 662 807

Potency (%) c

Abbreviations: Aib = 2-aminoisobutyric acid; Ab = 2-aminobutyric acid; Fmoc = fluorenyl methoxycarbonyl; Ahx = aminohexyl. a Guinea pig ileal strip contraction. b EDs0 ' half maximal concentration inducing ATP release from guinea pig platelets. c Threshold concentration for inducing aggregation of guinea pig platelets.

C-N-Y-I-T-E-L-R-R-Q-H-A-R-A-S-H-L-G-L-A-R W-W-G-S-K-L-G-L-A-R W-W-G-R-A-S-K-L-G-L-A-R W-W-G-Y-R-A-S-K-L-G-L-A-R W-W-G K-Y-R-A-S-K-L-G-L-A-R W-W-G-K-K-Y-R-A-S-K-L-G-L-A-R W-W-G-G-K-K-Y-R-A-S-K-L-G-L-A-R W-W-G-G-G-K-K-Y-R-A-S-K-L-G-L-A-R

C3a analogues

Ember et al., 1991

References

(b) Examples of potent synthetic analogues of human C3a

a

Lu et al., 1984 Kretzschmar et al., 1992 Lu et al., 1984 Ember et al., 1991 Hoeprich and Hugli, 1986 Hoeprich and Hugli, 1986 Gerardy-Schnhn et al., 1988; Ambrosius et al., 1989 Ember et al., 1991

Potency

2.0-4.3 nM 3 nM EDs0 b C-N Y-I-T-E-L-R-R-Q-H-A-R-A-S-H-L-G-L A - R (native sequence) 1 . 9 - 4 . 1 nM 92 nM c A-N-A-Aib-A-E-E-A-Aib-R-Q-A-Aib-R-A-A-Aib-L-G-L-A-R (helix-enhanced sequence) 1.2 nM A-N-A-Ab-A-P-A-Ab-R-Q-A-Ab-R-P-A-Ab-L-G-L-A-R (helix-disrupted sequence) 900 nM Fmoc-Ahx-Y-R-R-G-R-A-A-A-L-G-L-A-R 3 nM EDs0 b W-W-G-K-K-Y-R-A-S-K-L-G-L-A-R 6 nM c

Intact human C3a

C3a analogues

Table 2 (a) Examples of potent synthetic analogues of human C3a

I

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J.A. Ember, T.E. Hugli/lmmunopharmacology 38 (1997)3-15

sidue natural peptide C3a 57-77 in the guinea pig platelet aggregation assay (Lu et al., 1984; Ember et al., 1991). Based on these sequence and structural requirements for optimal biologic potency of a highly substituted C3a analogue, it appears that a bulky hydrophobic unit can specifically interact with certain residues at or near to the binding site on the C3a receptor. Current knowledge of the C3a conformation and structural requirements for these synthetic analogues provided a conceptual model for the C3a receptor-ligand interaction as shown in Fig. 3 (Ember et al., 1990). 3.2. C5a

Recent biologic studies, using synthetic C-terminal fragments of both human C5a (Chenoweth et al., 1979; Or et al., 1992; Ember et al., 1992; Morgan et al., 1992) and rat C5a (Carney and Hugli, 1993; Ember et al., 1993), provided evidence that the C-terminal portion of C5a indeed contains the effectot site of this molecule. Synthetic C-terminal analogues were prepared as models of the effector site in human C5a and were shown to exhibit the full spectrum of biological activities expressed by the parent protein. Active C5a analogues provided an opportunity to examine those parameters responsible for functional expression, including both the steric and conformational properties required for the effector portion of the ligand (Ember et al., 1992). It is known that removal of the C-terminal arginine from human C5a reduces spasmogenic potency by approximately 1000-fold, but only reduces the chemotactic activity by approximately 10-fold (Gerard et al.,

1979, Gerard and Hugli, 1981a,Gerard et al., 1981b). The C-terminal pentapeptide MQLGR (Chenoweth et al., 1979; Gerard et al., 1979) and the C-terminal octapeptide HKDMQLGR (Or et al., 1992; Kawai et al., 1992) were full agonists, with the exception that the synthetic fragments exhibit significantly lower potency (e.g. 0.01-0.1%) than the natural molecule. Efforts to design analogues with greater potency than those based on the natural C5a sequence resulted in highly modified C5a peptides of 8-10 residues in length (Table 3; Kawai et al., 1991a,b, 1992; Ember et al., 1992; Ki3hl et al., 1993). Major developments in these efforts were achieved by: (1) single residue replacements of the octamer HKDMQLGR, such as His 68 ~ Phe 68 (Or et al., 1992), that resulted in a 1000-1500-fold increase in receptor binding affinity; and (2) a systematic effort to increase hydrophobicity or alter chirality at selected positions that resulted in modified peptides with as much as a 20-fold increase in receptor affinity and biologic potency (Kawai et al., 1992; Siciliano et al., 1994). It has been suggested that regions other than the C-terminal portion of C5a, such as the N-terminal helical portion, either participate directly in receptor binding or in stabilizing binding sites elsewhere in the native C5a conformation (Gerard et al., 1985; Mollison et al., 1989). Dependence on secondary binding sites distant from the C-terminal region may explain why the intact factor is considerably more potent than synthetic C-terminal C5a analogues. Therefore, optimization of potency will require a more complex peptide design than simply constructing mimics of the C-terminal region. The conclusions based on C5a analogue studies suggest a two-

Table 3 Examples of potent synthetic analogues of human C5a C5a Analogues: Intact human C5a H-K-D-M-Q-L-G-R Y-S-F- K-P-M-P-L-(dA)-R H-K-D-Cha-Cha-L-(dA)-R F-K-D-M-Q-L-G-R F-L-A-Cha-Cha-(dA)-R Y-F-K-A-Cha-Cha-L-(dF)-R

Comments

Potency ~

References

(native sequence) ( H 67 ~ F + decreased flexibility) (increased hydrophobicity) ( s i n g l e H 67 ~ F substitution) (H 67 ---)F + increased hydrophobicity) (H 67 ~ F + increased hydrophobicity)

0.03 nM, K i 300/zM, K i 7.1 p~M, K i b 1.6 /zM, K i 0.2 /xM, K i 90 nM, K i 8 nM, K i

Or et al., 1992 Kawai et al., 1991a,b Tempero et al., 1997 Kawai et al., 1992 Oret al., 1992 Kawai et al., 1991 a Siciliano et al., 1994

Abbreviations: Cha = cyclohexylalanine; NMe = N-methyl; (dA), (dF) and (dR) are D-amino acids. a Ki determined by competition of human tZSl-C5a binding to human PMN membranes. b Ki determined by competition of human 1251-C5a binding to human PMNs.

J.A. Ember, T.E. Hugli / lmmunopharmacology 38 (1997)3-15

binding site model for ligand/receptor interactions for C5a, where the two binding sites appear to be separated from each other (i.e. non-contiguous). The anaphylatoxin receptor studies described below have greatly benefited from advances in peptide modeling of C5a analogue peptides.

4. Anaphylatoxin receptors Cloning and the molecular elucidation of anaphylatoxin receptors have given life to a new era in this field of research. There is now an opportunity to explore the ligand/receptor interactions in detail and to describe the nature of binding sites and structural elements that are essential in mediating cellular activation. The wide cellular distribution of these anaphylatoxin receptors and the many variable effects being identified suggest that perhaps a more prominent role is being played by these humoral factors than was recognized before this information was made available. We will describe the C5aR first in our discussion, because cloning and characterization of the C5aR preceded and greatly aided in the search for the C3aR, which had eluded identification and characterization until very recently. It is now clear that these two receptors are distinct molecules having quite separate physiologic roles to play. 4.1. C5aR

Early characterizations of the C5a receptor (CD88, C5aR) on human neutrophils clearly established three facts that later played a prominent role in elucidation of the C5a/C5aR interaction and on isolation of the receptor gene. It was determined that: (1) C5a (125I labeled) binds to a receptor on the neutrophil with nanomolar affinity; (2) there are as many as 105 copies of the receptor per cell; (3) and certain degradation products of C5a (i.e. C5ade S Arg and C5a 1-69) compete in binding with the intact factor (Chenoweth and Hugli, 1978). The observation that a C5a fragment, devoid of the C-terminal effector site (i.e. C5a 1-69), still binds to the receptor led to the hypothesis that both a primary effector and secondary binding site(s) exist on the C5a ligand and each are important for optimizing interaction with the receptor (Chenoweth and Hugli, 1978; Gerard et

9

al., 1979; Chenoweth et al., 1980). Characterization of the binding affinity and numbers of receptors on neutrophils, later estimated to average 80,000 copies/cell, with an affinity of approximately 2 nM (Huey and Hugli, 1985; Huey et al., 1986), provided the critical information indicating that differentiated leukocytic cell lines are appropriate sources from which to isolate and clone the C5aR gene. The gene structure for the human C5aR was reported by two separate groups in 1991. One group used a library obtained from dibutyryl-cAMP-induced human myeloid U937 cells (Gerard and Gerard, 1991) and the other group used a dibutyrylcAMP-induced human myeloid HL-60 cell library (Boulay et al., 1991). Both groups obtained cDNA clones with open reading frames of 1050 base pairs coding for an identical protein of 350 amino acid residues with a calculated Mr of 39,320. The C5aR protein sequence deduced from the cDNA clones identified the characteristic structure of a member of the rhodopsin superfamily of receptors, otherwise known as GTP-binding protein-coupled receptors or 7-transmembrane spanning receptors. A single glycosylation site was located at Asn 5 of the first extracellular domain of C5aR. The presence of a N-linked oligosaccharide group presumably explains the difference in size between the nude protein of 39 kDa and the 40-48 kDa estimated for C5aR expressed on human leukocytes. The size of the natural C5aR was estimated using a variety of chemical cross-linking techniques to attach ~25I-C5a to the receptor on neutrophils (Johnson and Chenoweth, 1985; Rollins and Springer, 1985; Huey and Hugli, 1985). The effects of glycosylation of C5aR on binding of the ligand were explored by replacing Asn 5 with an Ala residue (Pease and Barker, 1993). When both the Ala 5-C5aR mutant and wild type C5aR molecules were expressed on CHO cells and compared, the dissociation constants were 20 and 13 nM, respectively. These results suggest that glycosylation of the C5aR has little influence on either ligand binding or C5ainduced functions. Cloning of the C5aR (Boulay et al., 1991; Gerard and Gerard, 1991) provided new opportunities for elucidating the requirements for ligand-receptor interactions between C5a and its receptor. Antibodies generated against peptides based on the extracellular loops of C5aR were used to confirm receptor expres-

J.A. Ember, T.E. Hugli / lmmunopharmacology 38 (1997) 3-15

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sion and to investigate ligand binding to the C5aR. Antibodies generated to peptides that mimic portions of the N-terminal extracellular region of C5aR (residues 9-29) have proven to be excellent markers of cells and tissues expressing the receptor (Haviland et al., 1995; Gasque et al., 1995; Buchner et al., 1995; Gasque et al., 1997). This immunoreagent also blocks C5a binding and cellular activation indicating potential binding site for the ligand (Oppermann et al., 1993; Morgan et al., 1993). Surprisingly, neither cell binding nor cellular activation by the synthetic Cterminal analogues of C5a were blocked by these antibodies (Ember et al., 1994; Morgan et al., 1993). These results strongly suggested that the N-terminal region of the receptor is not the primary effector binding site for C5a, but rather defines a secondary non-effector binding site (Morgan et al., 1993).

Extracellular

The importance of the N-terminal region of C5aR in ligand binding was further confirmed in a series of mutagenesis studies. Truncation of the N-terminal 1-22 residues in C5aR (Siciliano et al., 1994; DeMartino et al., 1994) abrogated C5a binding, but had no effect on binding of the bioactive C-terminal analogues of C5a (Siciliano et al., 1994; DeMartino et al., 1994). Point-mutations were used to convert the five aspartic acid residues present in the N-terminal region to alanine (i.e. Asp 10, 15, 16, 21, 27 -~ Ala) resulting in significant loss in binding affinity of C5a. These data indicated the critical role of aspartic acid residues in C5aR for ligand/receptor interactions (Mery and Boulay, 1994; DeMartino et al., 1994). Additional studies creating receptor chimeras between portions of the formyl-peptide receptor (FPR) and C5aR confirmed the role of the

Asp16

Intrac ,314

Fig. 2. Regions and residues of huC5aR that are involved in ligand binding are identified on the extracellular surface of the receptor. This figure summarizes a number of point mutations and protein-truncation experiments, as represented by the work of several groups. Point mutations: residues that were found to be important for function or binding ( 0 , black); residues that were tested and found to have only moderate effects (O, yellow) as measured by changes in ligand binding affinities (Mery and Boulay, 1994; DeMartino et al., 1994, 1995; Monk et al., 1995). Receptor-trUncation: Elimination of residues 2-22, (black brackets) reduced ligand binding by 600-fold, and deletion of the whole N-terminus including residues 2-30 (green brackets) virtually eliminated C5a binding. C5aR-Formyl-peptide-receptor chimeras: loop regions found to be involved in ligand binding are outline in blue, loop regions that were tested and found not to be involved in ligand binding are outlined in yellow (Pease et al., 1994; Mery and Boulay, 1994). The serines (green) in the C-terminal intracellular region of C5aR are proposed sites of phosphorylation (Giannini et al., 1995).

J.A. Ember, T.E. Hugli/ lmmunopharmacology 38 (1997) 3-15

N-terminal region for ligand-binding (Mery and Boulay, 1994) and localized another binding site of C5aR to the region containing the second and third extracellular loops (Pease et al., 1994). These results, summarized in Fig. 2 indicate that the primary or effector binding site of C5a is located on the Cterminal half of C5aR. Studies focusing on localizing the primary effector-binding site on C5aR have used point-mutation techniques. Replacement of Glu 199 and Arg 206 by alanine caused a major depression in binding, thus identified these residues as part of the effector binding site in C5aR (Monk et al., 1995; DeMartino et al., 1995). These results concluded that specific residues of the second intracellular loop and residues on the fifth intramembrane helix may participate in formation of the primary effector-binding site of C5aR (see Fig. 2). 4.2. C3aR

The C3a receptor was first demonstrated on guinea pig platelets using cross-linking techniques and was estimated to be 95-105 kDa (Fukuoka and Hugli, 1988). A later report confirmed the unusually large size for a G-protein coupled receptor (i.e. 83-114 kDa) which exhibits a diffuse band in gels suggesting a highly glycosylated protein (Gerardy-Schahn et al., 1989). These data raised the question of how the C3aR could be related to the C5aR and yet be so much larger in size. Part of the mystery was solved once the human C3aR was cloned as reported by three different laboratories (Roglic et al., 1996; Ames et al., 1996; Crass et al., 1996). It was determined that the amino acid portion of human C3aR had a Mr of 53,864 Da and contained an additional 144 residues in the second extracellular domain when compared to C5aR. Sensitivity of C3a-dependent mobilization of intracellular calcium to pertussis toxin in differentiated U937 cells and in human neutrophils indicated that the C3aR was a G-protein coupled receptor (Klos et al., 1992; Norgauer et al., 1993). As with the C5aR, the known biologic effects of C3a, and evidence that C3a stimulates or binds to only selected cell types, led investigators to use expression libraries from human neutrophils or from differentiated leukocytic cells lines (i.e. HL-60 and U937) for isolating C3aR cDNA. Since there are glycosylation sites both at the N-terminal end and in

11

the large second extracellular loop of the human C3aR molecule, it was proposed that C3aR has both a higher oligosaccharide content and a larger protein size than most other known rhodopsin receptors, including C5aR. When the C3aR sequence is compared with that of C5aR or fMLP receptors there was only a 37.5 and 34.3% identity, respectively. Human C3aR has been expressed in HEK-293 and RBL-2H3 cells in which competitive binding with hC3a was demonstrated. C3aR expressed in CHO cells exhibited C3a-induced hydrolysis of phosphoinositides which confirms that the expressed C3aR exhibits functional activity (Crass et al., 1996). The C3aR has now been cloned from several animals including mouse (Hsu et al., 1997; Tornetta et al., 1997), guinea pig and rat (Fukuoka and Hugli, 1997a,Fukuoka et al., 1997b). The patterns of identity between C3aR obtained from different species show relatively high levels in the N-terminal extracellular region and for the transmembrane segments. The second intracellular loop is also highly conserved, perhaps because the two cysteinyl residues participate in critical disulfide bonds. The large second extracellular loop has only modest homology while the C-terminal intracellular region, which contains the G-protein binding site, is highly conserved. Because the major unique feature for C3aR compared to other rhodopsin receptors is the unusually large second extracellular loop, this region may prove to be particularly important for binding the C3a molecule. It has been postulated that the large extracellular loop might contain some or all the structural determinants for C3a binding, consequently this region will be a focus of future studies. Generation of loop-deletion mutants and chimeric C3aR/C5aR receptors are in progress in several laboratories, and these techniques will hopefully provide answers for these questions.

5. Modeling of the anaphylatoxin receptors Based on our current knowledge models are proposed for illustrating the interactions between C3a or C5a and their respective receptors (Fig. 3). The design for this models was adapted from the

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J.A. Ember, T.E. Hugli / lmmunopharmacology 38 (1997) 3-15

C5a/C5aR model originally proposed by Chenoweth and Hugli (Chenoweth et al., 1980) and refined by Siciliano et al. (1994). The C3a molecule has at least two major binding sites. A non-effector binding site (Site 1) exists on C3a along the C-terminal helical region and either makes contact with the large extracellular loop (as shown here) or with other exposed regions of the receptor.(See Fig. 3, view A). Site 2 contains the C-terminal effector region of C3a, in-

A. C3a/C3aR C

Exterior ;ite 2

Cytoplasm (

tor

B. C5aJC5aR Site

Exterior ~ite 2

C~op~sm tor

cluding the sequence LGLAR, which is shown penetrating into the 'pore' formed by the seven transmembrane regions of C3aR. This model for C3a/C3aR interaction corresponds to an earlier model proposed based on the extensive evidence for a multi-site binding of C5a with its receptor (Chenoweth et al., 1980). Based on all of the existing results, a model was designed which proposes that interaction occurs between the aspartic side chains in the N-terminal portion of C5aR and the conserved, cationic Arg 40 (and possibly Arg 37 and Lys 12) in human C5a. A second interaction must occur between other anionic sites on C5aR, presumably located on one of the extracellular loop or transmembrane regions, and the conserved residues Arg 62, His 66, Lys 67 and Arg 74 of the human C5a molecule. The model for C5a interaction with C5aR indicates that the non-effector site (Site 1) on C5a binds to the N-terminal region of the C5a receptor while the C-terminal effector site (Site 2) of C5a penetrates the 'pore'. Based on this model (see Fig. 3B), initial contact between C5a and the N-terminal receptor site could effectively raise the local C5a concentration, thereby promoting a cooperative interaction with the primary effector-binding site, resulting in cellular activation (Siciliano et al., 1994).

Fig. 3. Models are proposed for illustrating the interactions between C3a or C5a and their respective receptors. The design for these models was adapted from the C 5 a / C 5 a R model proposed by Siciliano et al. (1994). Both C3aR and C5aR are G-protein coupled transmembrane receptors of the large rhodopsin family. A. The C3a molecule has at least two major binding sites. A non-effector binding site (Site 1) exists on the C-terminal helical region of the ligand and it either makes contact with the large extracellular loop of C3aR (as shown here) or with other exposed regions of the receptor, such as the N-terminal region. Site 2 contains the C-terminal effector region of C3a, including the sequence LGLAR, which is shown penetrating into the 'pore" formed by the seven transmembrane regions of C3aR. This model for C 3 a / C 3 a R interaction corresponds to a model originally proposed for multi-site binding of C5a with its receptor (Chenoweth et al., 1980), B. The model proposed for C5a interactions with C5aR indicate that the non-effector site (Site 1) on C5a binds to the N-terminal region of the C5a receptor, while the C-terminal effector site (Site 2) of C5a penetrates the 'pore' formed by the transmembrane regions.

J.A. Ember, T.E. Hugli / Immunopharmacology 38 (1997) 3-15

6. Summary In summary, recent advances in molecular cloning of anaphylatoxins and the anaphylatoxin receptors add new dimensions to our investigations and understanding of the molecular mechanisms involved in anaphylatoxin action. Combining knowledge accumulated from peptide modeling of the ligands with mutagenesis studies of these ligands and their receptors makes it possible to more accurately model interactive sites and understand the sequence of molecular interactions required for cellular activation. In addition, these new developments provide valuable tools for investigating, yet unknown, activities and cellular targets of the anaphylatoxin molecules.

References Ambrosius, D., Casaretto, M., Gerardy-Schahn, R., Saunders, D., Brandenburg, D., Zahn, H., 1989. Peptide analogues of the anaphylatoxin C3a; Syntheses and properties. Biol. Chem. Hoppe-Seyler 370, 217-227. Ames, R.S., Li, Y., Saran, H.M., Nuthulaganti, P., Foley, J.J., Ellis, C., Zeng, Z., Su, K., Jurewicz, A.J., Hertzberg, R.P., Bergsma, D.J., Kumar, C., 1996. Molecular cloning and characterization of the human anaphylatoxin C3a receptor. J. Biol. Chem. 271, 20231-20234. Amsterdam, E.A., Stahl, G.L., Pan, H.L., Rendig, S.V., Fletcher, M.P., Longhurst, J.C., 1995. Limitation of reperfusion injury by a monoclonal antibody to C5a during myocardial infarction in pigs. Am. J. Physiol. 268, H448-H457. Barnum, S.R., 1995. Complement biosynthesis in the central nervous system. Crit. Rev. Oral. Biol. Med. 6, 132-146. Boulay, F., Mery, L., Tardif, M., Brouchon, L., Vignalis, P., 1991. Expression cloning of a receptor for C5a anaphylatoxin on differentiated HL-60 cells. Biochemistry 30, 2993-2999. Buchner, R.R., Hugli, T.E., Ember, J.A., Morgan, E.L., 1995. Expression of functional receptors for human C5a anaphylatoxin (CD88) on the human hepatocellular carcinoma cell line HepG2. Stimulation of acute-phase protein-specific mRNA and protein synthesis by human C5a anaphylatoxin. J. Immunol. 155, 308-315. Caporale, L.H., Tippett, P.S., Erickson, B.W., Hugli, T.E., 1980. The active site of C3a anaphylatoxin. J. Biol. Chem. 255, 10758-10763. Carney, D.F., Hugli, T.E., 1993. Site-specific mutations in the N-terminal region of human C5a that affect interactions of C5a with the neutrophil C5a receptor. Protein Sci. 2, 13911399. Carroll, M.C., Fischer, M.B., 1997. Complement and the immune response. Curr. Opin. Immunol. 9, 64-69.

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Chenoweth, D.E., Hugli, T.E., 1978. Demonstration of specific C5a receptor on intact human polymorphonuclear leukocytes. Proc. Natl. Acad. Sci. USA 75, 3943-3947. Chenoweth, D.E., Erickson, B.W., Hugli, T.E., 1979. Human C5a-related synthetic peptides as neutrophil chemotactic factors. Biochem. Biophys. Res. Commun. 86, 227-234. Chenoweth, D.E., Hugli, T.E., 1980. Human C5a and C5a analogs as probes of the neutrophil C5a receptor. Mol. lmmunol. 17, 151-161. Crass, T., Raffetseder, U., Martin, U., Grove, M., Klos, A., K~hl, J., Bautsch, W., 1996. Expression cloning of the human C3a anaphylatoxin receptor (C3aR) from differentiated U-937 cells. Eur. J. lmmunol. 26, 1944-1950. DeMartino, J.A., Van Riper, G., Siciliano, S.J., Molineaux, C.J., Konteastis, Z.D., Rosen, H., Springer, M.S., 1994. The amino terminus of the human C5a receptor is required for high affinity C5a binding and for receptor activation by C5a but not C5a analogs. J. Biol. Chem. 269, 14446-14450. DeMartino, J.A., Konteatis, Z.D., Siciliano, S.J., Van Riper, G., Underwood, D.J., Fischer, P.A., Springer, M.S., 1995. Arginine 206 of the C5a receptor is critical for ligand recognition and receptor activation by C-terminal hexapeptide analogs. J. Biol. Chem. 270, 15966-15969. Ember, J.A., Johansen, N.L., Hugli, T.E., 1990. A new approach to designing active analogues of proteins. Biochem. Soc. Trans. 18, 1154-1155. Ember, J.A., Johansen, N.L., Hugli, T.E., 1991. Designing synthetic superagonists of C3a anaphylatoxin. Biochemistry 30, 3603-3612. Ember, J.A., Sanderson, S.D., Taylor, S.M., Kawahara, M., Hugli, T.E., 1992. Biologic activity of synthetic analogues of C5a anaphylatoxin. J. lmmunol. 148, 3165-3173. Ember, J.A., Cui, L., Carney, D.F., Pettis, R.J., Erickson, B.W., Hugli, T.E., 1993. Molecular and functional characterization of superpotent rat C5a. Protein Sci. 2 (suppl. 1), 159, abstract. Ember, J.A., Morgan, E.L., Sanderson, S.D., Hugli, T.E., 1994. Anti-C5a receptor antibody inhibits biologic activity of C5a but not synthetic C5a analogues. FASEB J. 8, A500, abstract. Farries, T., Atkinson, J.P., 1991. Evolution of the complement system. Immunol. Today 12, 295-300. Foreman, K.E., Vaporciyan, A.A., Bonish, B.K., Jones, M.L., Glovsky, M.M., Eddy, S.M., Ward, P.A., 1994. C5a-induced expression of P-selectin in endothelial cells. J. Clin. Invest. 94, 1147-1155.

Frank, M.M., 1987. Complement in the pathophysiology of human disease. N. Engl. J. Med. 316, 1525-1530. Frank, M.M., 1989. Complement: a brief review. J. Allergy Clin. Immunol. 84, 411-420. Fukuoka, Y., Hugli, T.E., 1988. Demonstration of a specific C3a receptor on guinea pig platelets. J. Immunol. 140, 3496-3501. Fukuoka, Y., Hugli, T.E., 1997a. Molecular characterization of the guinea pig and rat C3a receptor. Microcirculation 4, 121, abstract. Fukuoka, Y., Ember, J.A., Hugli, T.E., 1997b. Molecular cloning of two isoforms of the guinea pig C3a receptor. J. Biol. Chem. (submitted). Gardinali, M., Conciato, L., Cafaro, C., Agostoni, A., 1995.

14

J.A. Ember, T.E. Hugli / lmmunopharmacology 38 (1997)3-15

Complement system in coronary heart disease: a review. Immunopharmacology 30, 105-117. Gasque, P., Chan, P., Fontaine, M., Ischenko, A., Lamacz. M., Giftze, O., Morgan, B.P., 1995. Identification and characterization of the complement C5a anaphylatoxin receptor on human astrocytes. Relevance to inflammation in the brain. J. Immunol. 155, 4882-4889. Gasque, P., Singhrao, S.K., Neal, J.W., G~Stze, O., Morgan, B.P., 1997. Expression of the receptor for C5a (CD88) is upregulated on reactive astrocytes, microglia and endothelial cells in inflamed human CNS. Am. J. Pathol. 150, 31-41. Gerard, C., Chenoweth, D.E., Hugli, T.E., 1979. Molecular aspects of the serum chemotactic factors. J. Reticuloendothel. Soc. 26, 711-718. Gerard, C., Hugli, T.E., 1981a. Identification of classical anaphylatoxin as the des-Arg form of the C5a molecule: evidence of a modulator role for the oligosaccharide unit in human desArg74-C5a. Proc. Natl. Acad. Sci. USA 78, 1833-1837. Gerard, C., Chenoweth, D.E., Hugli, T.E., 1981b. Response of human neutrophils to C5a: a role for the oligosaccharide moiety of human C5a-des Arg-74 but not of C5a in biologic activity. J. Immunol. 127, 1978-1982. Gerard, C., Showell, H.J., Hoeprich, P.D., Hugli, T.E., Stimler, N.P., 1985. Evidence for a role of the amino terminal region in the biological activity of the classical anaphylatoxin porcine C5a des Arg-74. J. Biol. Chem. 260, 2613-2616. Gerard, N.P., Gerard, C., 1991. The chemotactic receptor for human C5a anaphylatoxin. Nature 349, 614-617. Gerardy-Schahn, R., Ambrosius, D., Casaretto, M., Gr6tzinger, J., Saunders, D., Wollmer, A., Brandenburg, D.M,, Bitter-Suermann, D., 1988. Design and biological activity of a new generation of synthetic C3a analogues by combination of peptidic and non-peptidic elements. Biochem. J. 255,209-216. Gerardy-Schahn, R., Ambrosius, D., Saunders, D., Casaretto, M.. Mittler, C., Karwarth, G., Goergen, S., Bitter-Suermann, D.. 1989. Characterization of C3a receptor-proteins on guinea pig platelets and human polymorphonuclear leukocytes. Eur. J. Immunol. 19, 1095-1102. Giannini, E., Brouchon, L., Boulay, F., 1995. Identification of the major phosphorylation sites in human C5a anaphylatoxin receptor in vivo. J. Biol, Chem. 270, 19166-19172. Haviland, D.L., McCoy, R.L., Whitehead, W.T., Akama, H.. Molmenti, E.P., Brown, A., Haviland, J.C., Parks, W.C., 1995. Cellular expression of the C5a anaphylatoxin receptor (C5aR): Demonstration of C5aR on nonmyeloid cells of the liver and lung. J. Immunol. 154, 1861-1869. Hoeprich, P.D. Jr., Hugli, T.E., 1986. Helical conformation at the carboxy-terminal portion of human C3a is required for full activity. Biochemistry 25, 1945-1950. Hsu, M.H., Ember, J.A., Wang, M., Prossnitz, E.R., Hugli, T.E., Ye, R.D., 1997. Cloning and functional characterization of the mouse C3a anaphylatoxin receptor gene. Immunogenetics (in press). Huey, R., Hugli, T.E., 1985. Characterization of a C5a receptor on human polymorphonuclear leukocytes (PMN). J. lmmunol. 135, 2063-2068. Huey, R., Fukuoka, Y., Hoeprich, P.D. Jr., Hugli, T.E., 1986.

Cellular receptors to the anaphylatoxins C3a and C5a. Biochem. Soc. Syrup. 51, 69-81. Hugli, T.E., Erickson, B.W., 1977. Synthetic peptides with the biological activities and specificity of human C3a anaphylatoxin. Proc. Natl. Acad. Sci. USA 74, 1826-1830. lto~ B.R., Del Balzo, U., 1994. Effect of platelet depletion and inhibition of platelet cyclooxygenase on C5a-mediated myocardial iscbemia. Am. J. Physiol. 267, H1288-H1294. lvey, C.L., Williams, F.M., Collins, P.D., Jose, P.J., Williams, T.J., 1995. Neutrophil chemoattractaants generated in two phases during reperfusion of ischemic myocardium in the rabbit. Evidence for a role for C5a and interleukin-8 (comment). J. Clin. Invest. 95, 2720-2728. Johnston, R.B. Jr., 1993. The complement system in host defense and inflammation: the cutting edges of a double edged sword. Pediatr. Infect. Dis. 12, 933-941. Johnson, R.J., Chenoweth, D.E., 1985. Labeling the granulocyte C5a receptor with a unique photoreactive probe. J. Biol. Chem. 260, 7161-7164. Kalnik, M.W., Chazin, W.J., Wright, P.E., Villafranca, J.J. (Eds.), 1997. Techniques in Protein Chemistry II. The Three-Dimensional Solution Structure of Human Anaphylatoxin C3a. Academic Press, San Diego, CA, 37, pp. 393-400. Kawai, M., Lane, B., Mollison, K.W., Luly, J.R., Carter, G.W., 1991 a. Improved binding potency of a C5a related octapeptide by incorporation of N-methyl amino acids. 12th American Peptide Symp Cambridge, MA, #P-525 (abstract). Kawai, M., Quincy, D.A., Lane, B., Mollison, K.W., Luly, J.R., Carter, G.W., 1991b. Identification and synthesis of a receptor binding site of human anaphylatoxin C5a. J. Med. Chem. 34, 2068-2071. Kawai, M., Quincy, D.A., Lane, B., Mollison, K.W., Or, Y.S., Luly, J.R., Carter, G.W., 1992. Structure-function studies in a series of carboxyl-terminal octapeptide analogues of anaphylatoxin C5a. J. Med. Chem. 35, 220-223. Kazatchkine, M.D., Carreno, M.P., 1988. Activation of the complement system at the interface between blood and artificial surfaces. Biomaterials 9, 30-35. Kinoshita, T., 1991. Biology of complement: the overture. Immunol. Today 12, 291-295. Klos, A., Bank, S., Gietz, C., Bautsch, W., KiShl, J., Burg, M., Kretzschmar, T., 1992. C3a receptor on dibutyryl-cAMP-differentiated U937 cells and human neutrophils: The human C3a receptor characterized by functional responses and 125I-C3a binding. Biochemistry 31, 11274-11282. Kola, A., Klos, A., Bautsch, W., Kretzschmar, T., K~ihl, J., 1992. Functional activities of synthetic anaphylatoxic peptides in widely used biological assays. Clin. Exp. Immunol. 88, 368372. KShl, J., Casaretto, M., Gier, M., Karwath, G., Gietz, C., Bautsch, W., Saunders, D., Bitter-Suermann, D., 1990. Reevaluation of the C3a active site using short synthetic C3a analogues. Eur. J. Immunol. 20, 1463-1468. K~Shl, J., Liibbers, B., Klos, A , Bautsch, W., Casaretto, M., 1993. Evaluation of the C-terminal C5a effector site with short synthetic C5a analog peptides. Eur. J. Immunol. 23, 646-652. Kretzschmar, T., Pohl, M., Casaretto, M., Przewosny, M., Bautsch,

J.A. Ember, T.E. Hugli / Immunopharmacology 38 (1997) 3-15 W., Klos, A., Saunders, D., K~ihl, J., 1992. Synthetic peptides as antagonists of the anaphylatoxin C3a. Eur. J. Biochem. 210, 185-191. Lachmann, P.J., 1990. Protection against complement lysis. Biochem. Soc. Trans. 18, 1159-1160. Lu, Z.X., Fok, K.-F., Erickson, B.W., Hugli, T.E., 1984. Conformational analysis of COOH-terminal segments of human C3a. Evidence of ordered conformation in an active 21-residue peptide. J. Biol. Chem. 259, 7367-7370. Mery, L., Boulay, F., 1994. The NH2-terminal region of C5aR but not that of FPR is critical for both protein transport and ligand binding. J. Biol. Chem. 269, 3457-3463. Moffitt, M.C., Frank, M.M., 1994. Complement resistance in microbes. Springer Semin. Immunopathol. 15, 327-344. Mollison, K.W., Mandecki, W., Zuiderweg, E.R.P., Fayer, L., Fey, T.A., Krause, R.A., Conway, R.G., Miller, R.G., Edalji, L., Shallcross, M.A., Lane, B., Fox, J.L., Greer, J., Carter, G.W., 1989. Identification of receptor-binding residues in the inflammatory complement protein C5a site-directed mutagenesis. Proc. Natl. Acad. Sci. USA 86, 292. Monk, P.N., Barker, M.D., Partridge, L.J., Pease, J.E., 1995. Mutation of glutamate 199 of the human C5a receptor defines a binding site for ligand distinct from the receptor N terminus. J. Biol. Chem. 270, 16625-16629. Morgan, E.L., Sanderson, S., Scholz, W., Noonan, D.J., Weigle, W.O., Hugli, T.E., 1992. Identification and characterization of the effector region within human C5a responsible for stimulation of IL-6 synthesis. J. Immunol. 148, 3937-3942. Morgan, E.L., Ember, J.A., Sanderson, S.D., Scholz, W., Buchner, R., Ye, R.D., Hugli, T.E., 1993. Anti-C5a Receptor Antibodies. Characterization of neutralizing antibodies specific for a peptide, C5aR-(9-29), derived from the predicted amino-terminal sequence of the human C5a receptor. J. Immunol. 15l, 377-388. Mulligan, M.S., Schmid, E., Till, G.O., Friedl, H.P., Hugli, T.E., Johnson, K.J., Ward, P.A., 1996. Requirement and role of C5a in acute lung inflammatory injury in rats. J. Clin. Invest. 98, 503 -512. Mulligan, M.S., Schmid, E., Till, G.O., Hugli, T.E., Friedl, H.P., Roth, R.A., Ward, P.A., 1997. C5a-dependent upregulation in vivo of lung vascular P-selectin. J. Immunol. 158, 1857-1861. Miiller-Eberhard, H.J., 1988. Molecular organization and function of the complement system. Annu. Rev. Biochem. 57, 321-347. Norgauer, J., Dobos, G., Kownatzki, E., Dahinden, C., Burger, R., Kupper, R., Gierschik, P., 1993. Complement fragment C3a stimulates Ca 2+ influx in neutrophils via a pertussis-toxinsensitive G protein. Eur. J. Biochem. 217, 289-294.

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Oppermann, M., Raedt, U., Hebell, T., Schmidt, B., Zimmermann, B., Gtitze, O., 1993. Probing the human receptor for C5a anaphylatoxin with site-directed antibodies: Identification of a potential ligand binding site on the NH-2-terminal domain. J. Immunol. 151, 3785-3794. Or, Y.S., Clark, B., Lane, B., Mollison, K.W., Carter, G.W., Luly, J.R., 1992. Improvements in the minimum binding sequence of C5a: examination of His 67. J. Med. Chem. 35, 402-406. Pease, J.E., Barker, M.D., 1993. N-linked glycosylation of the C5a receptor. Biochemistry 31,719-726. Pease, J.E., Burton, D.R., Barker, M.D., 1994. Generation of chimeric C5a/formyl peptide receptors: towards the identification of the human C5a receptor binding site. Eur. J. Immunol. 24, 211-215. Reid, K.B., 1986. Activation and control of the complement system. Essays Biochem. 22, 27-68. Roglic, A., Prossnitz, E.R., Cavanagh, S.L., Pan, Z., Zou, A., Ye, R.D., 1996. cDNA cloning of a novel G protein-coupled receptor with a large extracellular loop structure. Biochim. Biophys. Acta 1305, 39-43. Rollins, T.E., Springer, M.S., 1985. Identification of the polymorphonuclear leukocyte C5a receptor. J. Biol. Chem. 260, 71577160. Rother, K., Rother, U., 1986. Biological functions of the complement system. Prog. Allergy 39, 24-100. Schmid, E., Warner, R.L., Crouch, L.D., Friedl, H.P., Till, G.O., Hugli, T.E., Ward, P.A., 1997. Neutrophil chemotactic activity and C5a following systemic activation of complement in rats. Inflammation (in press). Siciliano, S.J., Rollins, T.E., DeMartino, J., Konteatis, Z., Malkowitz, L., Van Riper, G., Bondy, S., Rosen, H., Springer, M.S., 1994. Two-site binding of C5a by its receptor: An alternative binding paradigm for G protein-coupled receptors. Proc. Natl. Acad. Sci. USA 91, 1214-1218. Tempero, R.M., Hollingsworth, M.A., Burdick, M.D., Finch, A.M., Taylor, S.M., Vogen, S.M., Morgan, E.L., Sanderson, S.D., 1997. Molecular adjuvant effects of a conformationally biased agonist of human C5a anaphylatoxin. J. Immunol. 158, 1377-1382. Tornetta, M.A., Foley, J.J., Saran, H.M., Ames, R.S., 1997. The mouse anaphylatoxin C3a receptor: molecular cloning, genomic organization and functional expression. J. Immunol. 158, 5277-5282. Ward, P.A., 1996. Rous-Whipple Award Lecture. Role of complement in lung inflammatory injury. Am. J. Pathol. 149, 10811086.