Structural and functional characterization of the human formyl peptide receptor ligand-binding region

Abstract

The formyl peptide (N-formyl-1-methionyl-1-leucyl-1-phenylalanine [FMLP]) receptor is involved in the activation of neutrophils and their subsequent response to chemotactic N-formylated peptides. Recently, we found that the first extracellular loop closest to the N-terminal end of the FMLP receptor exhibited the strongest ligand binding compared with that shown by other extracellular regions. By constructing amino acid substitutional variants of this domain, we have determined that residues Arg-84 and Lys-85 on this loop play major roles in ligand-binding activity. Furthermore, random rearrangement of the residues of this receptor region demonstrated that the position of these charged amino acids did not affect their involvement in ligand binding, although their presence was essential for this binding to occur. We propose that the portion of the first N-terminal extracellular loop of the FMLP receptor containing residues Arg-84 and Lys-85 contributes significantly to the active site in ligand-receptor binding. We further propose that this binding is not dependent on defined structure but rather that these charged moieties may function as important "contacts" in receptor-ligand interactions.Polymorphonuclear leukocytes (neutrophils) provide an important host defensive mechanism against bacterial and fungal infections. Neutrophils respond to inflammation both in vitro and in vivo through a chemotactic pathway of directed locomotion towards a stimulus (22). N-formylated peptides such as N-formyl-1-methionyl-1-leucyl-1-phenylalanine (FMLP), believed to be structural analogs of bacterial metabolic products, are potent chemoattractants for neutrophils (26,33). In addition to chemotaxis, a number of neutrophil functions, including aggregation, phagocytosis, generation of cytotoxic superoxide anions, production of bioactive arachidonate metabolites, and proteolytic enzyme release, can be elicited by potent chemoattractants such as FMLP (3,6,23,28,32). Through the use of radioiodinated cross-linking reagents and photoaffinity labeling techniques, the human neutrophil FMLP receptor has been reported to consist of three components, the predominant glycoprotein having a molecular weight of 55,000 to 70,000 (12,17,25). Functional responses of neutrophils are the result of ligand binding to specific plasma membrane receptors and appear to be mediated through a transduction mechanism involving the activation of phospholipase C and specific guanine nucleotide-binding proteins (18), which are involved in the receptor-mediated control of neutrophil function (19). Recently, the human FMLP receptor has been proposed to be a seven-transmembrane-domain structure characteristic of the G-protein-coupled receptor superfamily (7,8).The production of synthetic peptides from the known amino acid sequence of a biologically active protein allows the characterization of the immunological and functional domains of that protein. This technique has been successfully utilized in the identification of functional domains of receptors such as the pho...

Human formyl peptide receptor function

“…A similar decrease (33‐fold) was observed when Lys85 was mutated to alanine; however, the extent of reduction was less than that observed with the Arg84 mutation. These two residues (Arg84 and Lys85) are located in the N‐terminal region of the first extracellular loop, thereby reconfirming previous synthetic peptide studies from our laboratory [17]. Mutating Asp284 to alanine resulted in a 23‐fold reduction in the binding affinity of the receptor.…”

“…Mutating Asp284 to alanine resulted in a 23‐fold reduction in the binding affinity of the receptor. Asp284 is located in the C‐terminal region of the fifth extracellular loop of FPR, another region which has been implicated in ligand binding by peptide inhibition studies [17]. Mutating Arg205 and Arg163 also reduced the binding affinity by 58‐fold and 28‐fold, respectively.…”

Molecular recognition of peptide and non-peptide ligands by the extracellular domains of neurohypophysial hormone receptors

“…It is also suggested that part of the N-terminal domain, together with all of the first and part of the second extracellular loops, are spatially orientated so as to provide a ligand-recognition site (Figure 3). It is possible that the N-terminal subdomain forms a ' lid ' over the top of the ligand-occupied receptor as has been suggested for the formyl peptide receptor [27]. Such interaction with receptor extracellular domains has now been demonstrated for a variety of peptide ligands, from small formyl peptides to large glycoproteins [28][29][30][31][32][33][34][35].…”

The Molecular Basis for Neutrophil Dysfunction in Early‐Onset Periodontitis

“…These charged moieties may function as important contacts in receptor-ligand interactions. 81 We have confirmed this by recombinant expression of a truncated FMLP receptor mutant.82 However, complete FMLP binding to the receptor appears to involve the other extracellular loops. 80 Multiple areas of ligand binding of the FMLP receptor have also been suggested by others.…”

“…[83][84] Likewise, activation of G proteins also requires the interaction of multiple intracellular portions of the receptor molecule with the G proteins themselves. [76][77][78][79][80][81][82][83][84][85] Interestingly enough, the second intracellular loop of the receptor molecule appears to play the major role in receptor-G protein interactions.76 Figure 1 depicts a diagram of the receptor.…”