MinireviewThe human formyl peptide receptor as model system for constitutively active G-protein-coupled receptors
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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 Gi-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 Gi-proteins (Fig. 4A), and on Na+, stabilizing the R state of the FPR (Fig. 4C).
In
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 Gi-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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2012, Biochemical PharmacologyCitation Excerpt :The formyl peptide N-formyl-l-methionyl-l-leucyl-l-phenylalanine (fMLP) is a bacterial by-product of protein synthesis and plays an important role in inflammatory disorders [11,12]. fMLP binds to Gi-protein-coupled formyl-peptide receptors resulting in activation of both neutrophil and eosinophil granulocytes [11,12]. Histamine, a biogenic amine which is generated by decarboxylation of the amino acid l-histidine, is released from mast cells and basophils and serves as an autacoid and neurotransmitter [13].
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2010, Pharmacology and TherapeuticsCitation Excerpt :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).