The human formyl peptide receptor as model system for constitutively active G-protein-coupled receptors

doi:10.1016/S0024-3205(03)00654-4

Life Sciences

Volume 73, Issue 18, 19 September 2003, Pages 2263-2280

https://doi.org/10.1016/S0024-3205(03)00654-4 Get rights and content

Abstract

According to the two-state model of G-protein-coupled receptor (GPCR) activation, GPCRs isomerize from an inactive (R) state to an active (R*) state. In the R* state, GPCRs activate G-proteins. Agonist-independent R/R* isomerization is referred to as constitutive activity and results in an increase in basal G-protein activity, i.e. GDP/GTP exchange. Agonists stabilize the R* state and further increase, whereas inverse agonists stabilize the R state and decrease, basal G-protein activity. Constitutive activity is observed in numerous wild-type GPCRs and disease-causing GPCR mutants with increased constitutive activity. The human formyl peptide receptor (FPR) exists in several isoforms (FPR-26, FPR-98 and FPR-G6) and activates chemotaxis and cytotoxic cell functions of phagocytes through G_i-proteins. Studies in HL-60 leukemia cell membranes demonstrated inhibitory effects of Na⁺ and pertussis toxin on basal G_i-protein activity, suggesting that the FPR is constitutively active. However, since HL-60 cells express several constitutively active chemoattractant receptors, analysis of constitutive FPR activity was difficult. Sf9 insect cells do not express chemoattractant receptors and G_i-proteins and provide a sensitive reconstitution system for FPR/G_i-protein coupling. Such expression studies showed that FPR-26 is much more constitutively active than FPR-98 and FPR-G6 as assessed by the relative inhibitory effects of Na⁺ and of the inverse agonist cyclosporin H on basal G_i-protein activity. Site-directed mutagenesis studies suggest that the E346A exchange in the C-terminus critically determines dimerization and constitutive activity of FPR. Moreover, N-glycosylation of the N-terminus seems to be important for constitutive FPR activity. Finally, we discuss some future directions of research.

Section snippets

Introduction: constitutive GPCR activity

GPCRs constitute the largest gene family in the human genome and possess seven transmembrane domains (TM1-TM7), three extracellular loops (e1-e3), three intracellular loops (i1-i3), an extracellularly located N-terminus and an intracellularly located C-terminus Bockaert and Pin, 1999, Gudermann et al., 1997. GPCRs mediate signal transduction of intercellular signaling molecules (hormones, neurotransmitters, autacoids). Intercellular signaling molecules are agonists at GPCRs. Upon binding of

(Patho)physiological roles of the FPR

The FPR represents a chemoattractant receptor that is expressed in professional phagocytes, e.g., neutrophils, basophils and monocytes Dillon et al., 1988, Le et al., 2002a, Murphy, 1994, Prossnitz and Ye, 1997, Seifert and Schultz, 1991, Wenzel-Seifert and Seifert, 2001. The FPR is activated by formyl peptides released from bacteria Marasco et al., 1984, Schiffmann et al., 1975. Phagocytes migrate along a concentration gradient towards the site of formyl peptide production, a process referred

G-protein coupling of the FPR

G-proteins are heterotrimeric and consist of a α-subunit and a βγ-complex Birnbaumer et al., 1990, Gilman, 1987. Physiologically, the FPR is predominantly if not exclusively coupled to pertussis toxin (PTX)-sensitive G_i-proteins Dillon et al., 1988, Klinker et al., 1996, Seifert and Schultz, 1991, Wenzel-Seifert and Seifert, 2001, although in recombinant systems, the FPR also possesses the potential to couple to the PTX-insensitive G-proteins Gα₁₅ and Gα₁₆ (Offermanns and Simon, 1995).

Early evidence for constitutive FPR activity

Detailed analysis of constitutive GPCR activity critically depends on the availability of inverse agonists that reduce basal G-protein activity (Seifert and Wenzel-Seifert, 2002). However, until about 5 years ago (Wenzel-Seifert et al., 1998), an inverse FPR agonist was not known. Therefore, early studies on constitutive FPR activity relied on the use of PTX, blocking the interaction of the agonist-free FPR with G_i-proteins (Fig. 4A), and on Na⁺, stabilizing the R state of the FPR (Fig. 4C).

Uncovering constitutive activity of FPR-26 in Sf9 insect cells

Based on the above-discussed data, a sensitive recombinant expression system had to be developed in order to study a defined chemoattractant receptor without interference of other potentially constitutively active GPCRs. We decided to express chemoattractant receptors and G_i-proteins in Spodoptera frugiperda (Sf9) insect cells for several reasons. First, these cells can be easily cultured in spinner cultures, allowing for the preparation of large amounts of membranes (Gether et al., 1995).

Molecular determinants of the high constitutive activity of FPR-26

Our data regarding the high constitutive activity of the complement C5a receptor did not rule out the possibility than the presence of FPR isoforms with low constitutive activity contributed to, or even fully explained, the lack of inverse agonistic activity of CsH in HL-60 membranes. Therefore, we compared the constitutive activity of the FPR isoforms 26, 98 and G6. In fact, FPR-98 and FPR-G6 exhibited very different biochemical properties than FPR-26 including constitutive activity

Conclusions and some future studies

Analysis of the FPR highlighted the value of recombinant systems to study constitutive GPCR activity. Those studies revealed that closely related members of a GPCR family, i.e. chemoattractant receptors, exhibit largely different constitutive activity. The non-availability of inverse agonists is not detrimental for the analysis of constitutive activity of chemoattractant receptors because Na⁺ can be used as universal stabilizer of the R state. Strikingly, even naturally occurring FPR isoforms

Acknowledgements

This work was supported by the National Institutes of Health COBRE award 1 P20 RR15563 and matching support from The State of Kansas and The University of Kansas, and a grant from the Army Research Office (DAAD 19-00-1-0069).

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To this end, however, the (patho)physiological relevance of the differences between the polymorphic GPCR forms FPR-26, FPR-98 and FPR-G6 observed at a molecular level in Sf9 insect cell membranes is still elusive. Taken together, FPR can be regarded as a model system for constitutive activity of GPCRs, and the detailed investigation of this phenomenon at the FPR would hardly have been possible without the use of the Sf9 cell/baculovirus expression system (Seifert & Wenzel-Seifert, 2003). Two homologues of the murine CXCR4 were reconstituted with Gαi2 and Gβ1γ3 in baculovirus-infected insect cells, resulting in SDF-1α-mediated activation of [35S]GTPγS binding to Gαi2 (Moepps et al., 1997).
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MinireviewThe human formyl peptide receptor as model system for constitutively active G-protein-coupled receptors

Abstract

Section snippets

Introduction: constitutive GPCR activity

(Patho)physiological roles of the FPR

G-protein coupling of the FPR

Early evidence for constitutive FPR activity

Uncovering constitutive activity of FPR-26 in Sf9 insect cells

Molecular determinants of the high constitutive activity of FPR-26

Conclusions and some future studies

Acknowledgements

Biochimica Biophysica Acta

Journal of Biological Chemistry

Biochemical Pharmacology

Biochimica Biophysica Acta

European Journal of Pharmacology

Journal of Allergy and Clinical Immunology

Journal of Biological Chemistry

Journal of Biological Chemistry

European Journal of Pharmacology

Journal of Biological Chemistry

Journal of Biological Chemistry

General Pharmacology

Biochemical Pharmacology

Trends in Pharmacological Sciences

Trends in Immunology

International Immunopharmacology

Trends in Pharmacological Sciences

Trends in Pharmacological Sciences

Journal of Biological Chemistry

Journal of Biological Chemistry

Journal of Biological Chemistry

Gene

FEBS Letters

Journal of Biological Chemistry

Journal of Biological Chemistry

Journal of Biological Chemistry

Pharmacology and Therapy

Journal of Biological Chemistry

Journal of Biological Chemistry

Journal of Biological Chemistry

Biochemical and Biophysical Research Communications

Broad immunocytochemical localization of the formylpeptide receptor in human organs, tissues, and cells

Cell Tissue Research

A conserved tyrosine residue (Y601) in transmembrane domain 5 of the human thyrotropin receptor serves as a molecular switch to determine G-protein coupling

FASEB Journal

Molecular tinkering of G protein-coupled receptors: an evolutionary success

EMBO Journal

Physiological effects of inverse agonists in transgenic mice with myocardial overexpression of the β2-adrenoceptor

Nature

The human N-formylpeptide receptor. Characterization of two cDNA isolates and evidence for a new subfamily of G-protein-coupled receptors

Biochemistry

Pertussis toxin inhibits chemotactic peptide-stimulated generation of inositol phosphates and lysosomal enzyme secretion in human leukemic (HL-60) cells

Proceedings of the National Academy of Sciences of the United States of America

Pharmacology of muscarinic receptor subtypes constitutively activated by G proteins

Molecular Pharmacology

Isozyme-selective stimulation of phospholipase C-β2 by G protein βγ-subunits

Nature

Mitochondrial N-formylmethionyl proteins as chemoattractants for neutrophils

Journal of Experimental Medicine

Characterization of chenodeoxycholic acid as an endogenous antagonist of the G-coupled formyl peptide receptors

Inflammation Research

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Molecular Pharmacology

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The acute inflammatory response and the role of phagocytic cells in periodontal health and disease

Periodontology

Selective inhibition of N-formylpeptide-induced neutrophil activation by carbamate-modified peptide analogues

Biochemistry

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Virchows Archives B of Cellular Pathology Including Molecular Pathology

Constitutive activity of the human β1-adrenergic receptor in β1-receptor transgenic mice

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Minireview
The human formyl peptide receptor as model system for constitutively active G-protein-coupled receptors

Physiological effects of inverse agonists in transgenic mice with myocardial overexpression of the β₂-adrenoceptor

Isozyme-selective stimulation of phospholipase C-β₂ by G protein βγ-subunits

Constitutive activity of the human β₁-adrenergic receptor in β₁-receptor transgenic mice

Reconstitution of recombinant bovine A₁ adenosine receptors in Sf9 cell membranes with recombinant G proteins of defined composition