The combined presence of CD20 + B cells and PD-L1 + tumor-infiltrating lymphocytes in inflammatory breast cancer is prognostic of improved patient outcome.

PURPOSE: The purpose of the study was to evaluate protein expression of PD-L1 and CD20 as prognostic biomarkers of patient outcome in inflammatory breast cancer (IBC) samples. METHODS: PD-L1 and CD20 protein expression was measured by immunohistochemistry in 221 pretreatment IBC biopsies. PD-L1 was assessed in tumor cells (PD-L1+ tumor cells) and tumor stromal infiltrating lymphocytes (PD-L1+ TILs); CD20 was scored in tumor-infiltrating B cells. Kaplan-Meier curves and Cox proportional hazard models were used for survival analysis. RESULTS: PD-L1+ tumor cells, PD-L1+ TILs, and CD20+ TILs were found in 8%, 66%, and 62% of IBC, respectively. PD-L1+ tumor cells strongly correlated with high TILs, pathological complete response (pCR), CD20+ TILs, but marginally with breast cancer-specific survival (BCSS, P = 0.057). PD-L1+ TILs strongly correlated with high TILs, CD20+ TILs, and longer disease-free survival (DFS) in all IBC and in triple-negative (TN) IBC (P < 0.035). IBC and TN IBC patients with tumors containing both CD20+ TILs and PD-L1+ TILs (CD20+TILs/PD-L1+TILs) showed longer DFS and improved BCSS (P < 0.002) than patients lacking both, or those with either CD20+ TILs or PD-L1+ TILs alone. In multivariate analyses, CD20+TILs/PD-L1+TILs status was an independent prognostic factor for DFS in IBC (hazard ratio (HR): 0.53, 95% CI 0.37-0.77) and TN IBC (HR: 0.39 95% CI 0.17-0.88), and for BCSS in IBC (HR: 0.60 95% CI 0.43-0.85) and TN IBC (HR: 0.38 95% CI 0.17-0.83). CONCLUSION: CD20+TILs/PD-L1+TILs status represents an independent favorable prognostic factor in IBC and TN IBC, suggesting a critical role for B cells in antitumor immune responses. Anti-PD-1/PD-L1 and B cell-activating immunotherapies should be explored in these settings.

Obesity, insulin resistance and diabetes: sex differences and role of oestrogen receptors.

Obesity increases the risk of coronary artery disease through insulin resistance, diabetes, arterial hypertension and dyslipidemia. The prevalence of obesity has increased worldwide and is particularly high among middle-aged women and men. After menopause, women are at an increased risk to develop visceral obesity due to the loss of endogenous ovarian hormone production. Effects of oestrogens are classically mediated by the two nuclear oestrogen receptors (ERs) α and ß. In addition, more recent research has shown that the intracellular transmembrane G-protein-coupled oestrogen receptor (GPER) originally designated as GPR30 also mediates some of the actions attributed to oestrogens. Oestrogen and its receptors are important regulators of body weight and insulin sensitivity not only in women but also in men as demonstrated by ER mutations in rodents and humans. This article reviews the role of sex hormones and ERs in the context of obesity, insulin sensitivity and diabetes as well as the related clinical issues in women and men.

Extra-nuclear estrogen receptor GPR30 regulates serotonin function in rat hypothalamus.

Selective serotonin reuptake inhibitors (SSRIs), such as Prozac, are used to treat mood disorders. SSRIs attenuate (i.e. desensitize) serotonin 1A (5-HT(1A)) receptor signaling, as demonstrated in rats through decreased release of oxytocin and adrenocorticotropin hormone (ACTH) following 5-HT(1A) receptor stimulation. Maximal therapeutic effects of SSRIs for treatment of mood disorders, as well as effects on hypothalamic 5-HT(1A) receptor signaling in animals, take 1 to 2 weeks to develop. Estradiol also attenuates 5-HT(1A) receptor signaling, but, in rats, these effects occur within 2 days; thus, estrogens or selective estrogen receptor modulators may serve as useful short-term tools to accelerate desensitization of 5-HT(1A) receptors in response to SSRIs if candidate estrogen receptor targets in the hypothalamus are identified. We found high levels of GPR30, which has been identified recently as a pertussis-toxin (PTX) sensitive G-protein-coupled estrogen receptor, in the hypothalamic paraventricular nucleus (PVN) of rats. Double-label immunohistochemistry revealed that GPR30 co-localizes with 5-HT(1A) receptors, corticotrophin releasing factor (CRF) and oxytocin in neurons in the PVN. Pretreatment with PTX to the PVN before peripheral injections of 17-beta-estradiol 3-benzoate completely prevented the reduction of the oxytocin response to the 5-HT(1A) receptor agonist, (+)-8-hydroxy-2-dipropylaminotetralin (DPAT). Treatment with the selective GRP30 agonist, G-1, attenuated 5-HT(1A) receptor signaling in the PVN as measured by an attenuated oxytocin (by 29%) and ACTH (by 31%) response to DPAT. This study indicates that a putative extra-nuclear estrogen receptor, GPR30, may play a role in estradiol-mediated attenuation of 5-HT(1A) receptor signaling, and potentially in accelerating the effects of SSRIs in treatment of mood disorders.

Modulation of GPCR conformations by ligands, G-proteins, and arrestins.

G-protein-coupled receptors (GPCRs) have traditionally been thought to adopt two conformations: the inactive unliganded conformation and the active ligand-bound conformation. Interactions with G-proteins in cells and membranes are known to modulate the affinity of the receptor for ligand and therefore the conformation of the receptor. Such observations led to the proposal of the ternary complex model. However, subsequent studies of constitutively active GPCRs led to the development of an extended version of this model to account for active conformations of the receptor in the absence of agonist. A significant difficulty with many of the studies, upon which this latter model was based, is the lack of knowledge of receptor and G-protein concentrations due to the two-dimensional nature of the membranes used to perform the measurements. Over the past decade, we have studied the interaction of GPCRs, G-proteins, arrestins, and ligands in solubilized systems, where the concentration of each component can be defined. Here we summarize results of these studies as they pertain to the regulation of GPCR conformations and affinities for interacting species.

High throughput screening of G-protein coupled receptors via flow cytometry.

The molecular assemblies of signal transduction components, for example kinases and their target proteins or receptor-ligand complexes and intracellular signaling molecules, are critical for biological functions in cells. To better understand the interactions of these molecular assemblies and to screen for new pharmaceutics that could control and modulate these types of interactions, we have focused on developing high throughput approaches for the analysis of G-protein coupled receptors via flow cytometry. Flow cytometry offers a number of advantages including real-time collection of multicomponent data, and together with improvements in sample handling, the high throughput sampling rate is up to 100 samples per minute. For our targets, assemblies of solubilized GPCRs, a screening platform of a dextran bead has proven to be flexible, allowing different surface chemistries on the beads. The bead can be either ligand-labeled or have epitope-linked proteins attached to the bead surface, enabling several molecular assemblies to be constructed and analyzed. A major improvement with this system is that for screening ligands for GPCRs the underlying mechanism of action for these compounds can be investigated and incorporated into the definition of a 'hit'. Our current screening system is capable of simultaneously distinguishing GPCR agonists and antagonists.

HTPS flow cytometry: a novel platform for automated high throughput drug discovery and characterization.

The flow cytometer is unique among biomedical analysis instruments because it makes simultaneous and multiple optical measurements on individual cells or particles at high rates. High throughput flow cytometry represents a potentially important multifactorial approach for screening large combinatorial libraries of compounds. Limiting this approach has been the availability of instrumentation and methods in flow cytometry for automated sample handling on the scale required for drug discovery applications. Here, we describe an automated system in which a novel patented fluidics-based pharmacology platform, the HTPS (High Throughput Pharmacological System), is coupled to a flow cytometer using a recently described plug flow-coupling valve technology. Individual samples are aspirated sequentially from microplate wells and delivered to a flow cytometer for rapid multiparametric analysis. For primary screening to detect and quantify cell fluorescence in endpoint assays, a high-speed no-wash protocol enabled processing of 9-10 cell samples/min from 96-well microplates. In an alternate primary screening format, soluble receptor ligands were sampled from microplate wells at rates of 3-4 samples/minute and successfully assessed for the ability to elicit intracellular calcium responses. Experiments with fluorescent beads validated the accurate automated production by the HTPS of exponential and linear gradients of soluble compounds. This feature enabled rapid (2- to 3-min) characterization of the intracellular calcium dose response of myeloid cells to formyl peptide as well as the quantitative relationship between formyl peptide receptor occupancy and cell response. HTPS flow cytometry thus represents a powerful high throughput multifactorial approach to increase the efficiency with which novel bioresponse-modifying drugs may be identified and characterized.

Real time analysis of the affinity regulation of alpha 4-integrin. The physiologically activated receptor is intermediate in affinity between resting and Mn(2+) or antibody activation.

This work examines the affinity of alpha(4)beta(1)-integrin and whether affinity regulation by G protein-coupled receptor (GPCR) and chemokines receptors is compatible with cell adhesion mediated between alpha(4)-integrin and vascular cell adhesion molecule-1. We used flow cytometry to examine the binding of a fluorescent derivative of an LDV peptide (Chen, L. L., Whitty, A., Lobb, R. R., Adams, S. P., and Pepinsky, R. B. (1999) J. Biol. Chem. 274, 13167-13175) to several cell lines and leukocytes with alpha(4)-integrin ranging from about 2,000 to 100,000 sites/cell. The results support the idea that alpha(4)-integrins exhibit multiple affinities and that affinity changes are regulated by the dissociation rate and conformation. The affinity varies by 3 orders of magnitude with the affinity induced by binding mAb TS2/16 plus Mn(2+) > Mn(2+) ' TS2/16 > activation because of occupancy of GPCR or chemokines receptor > resting receptors. A significant fraction of the receptors respond to the activating process. The change in alpha(4)-integrin affinity and the corresponding change in off rates mediated by GPCR receptor activation are rapid and transient, and their duration depends on GPCR desensitization. The affinity changes mediated by IgE receptor or interleukin-5 receptor persist longer. It appears that the physiologically active state of the alpha(4)-integrin, determined by inside-out signaling, has similar affinity in several cell types.

Regulation of formyl peptide receptor agonist affinity by reconstitution with arrestins and heterotrimeric G proteins.

Although heptahelical chemoattractant and chemokine receptors are known to play a significant role in the host immune response and the pathophysiology of disease, the molecular mechanisms and transient macroassemblies underlying their activation and regulation remain largely uncharacterized. We report herein real time analyses of molecular assemblies involving the formyl peptide receptor (FPR), a well described member of the chemoattractant subfamily of G protein-coupled receptors (GPCRs), with both arrestins and heterotrimeric G proteins. In our system, the ability to define and discriminate distinct, in vitro receptor complexes relies on quantitative differences in the dissociation rate of a fluorescent agonist as well as the guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) sensitivity of the complex, as recently described for FPR-G protein interactions. In the current study, we demonstrate a concentration- and time-dependent reconstitution of liganded, phosphorylated FPR with exogenous arrestin-2 and -3 to form a high agonist affinity, nucleotide-insensitive complex with EC(50) values of 0.5 and 0.9 microm, respectively. In contrast, neither arrestin-2 nor arrestin-3 altered the ligand dissociation kinetics of activated, nonphosphorylated FPR. Moreover, we demonstrated that the addition of G proteins was unable to alter the ligand dissociation kinetics or induce a GTP gamma S-sensitive state of the phosphorylated FPR. The properties of the phosphorylated FPR were entirely reversible upon treatment of the receptor preparation with phosphatase. These results represent to our knowledge the first report of the reconstitution of a detergent-solubilized, phosphorylated GPCR with arrestins and, furthermore, the first demonstration that phosphorylation of a nonvisual GPCR is capable of efficiently blocking G protein binding in the absence of arrestin. The significance of these results with respect to receptor desensitization and internalization are discussed.

Partial phosphorylation of the N-formyl peptide receptor inhibits G protein association independent of arrestin binding.

It is now well accepted that G protein-coupled receptors activated by agonist binding become targets for phosphorylation, leading to desensitization of the receptor. Using a series of phosphorylation deficient mutants of the N-formyl peptide receptor (FPR), we have explored the role of phosphorylation on the ability of the receptor to interact with G proteins and arrestins. Using a fluorometric assay in conjunction with solubilized receptors, we demonstrate that phosphorylation of the wild type FPR lowers its affinity for G protein, whereas mutant receptors lacking four potential phosphorylation sites retain their ability to couple to G protein. Phosphorylated mutant receptors lacking only two potential phosphorylation sites are again unable to couple to G protein. Furthermore, whereas stimulated wild type FPR in whole cells colocalizes with arrestin-2, and the solubilized, phosphorylated FPR binds arrestin-2, the stimulated receptors lacking four potential phosphorylation sites display no interaction with arrestin-2. However, the mutant receptors lacking only two potential phosphorylation sites are restored in their ability to bind and colocalize with arrestin-2. Thus, there is a submaximal threshold of FPR phosphorylation that simultaneously results in an inhibition of G protein binding and an induction of arrestin binding. These results are the first to demonstrate that less than maximal levels of receptor phosphorylation can block G protein binding, independent of arrestin binding. We therefore propose that phosphorylation alone may be sufficient to desensitize the FPR in vivo, raising the possibility that for certain G protein-coupled receptors, desensitization may not be the primary function of arrestin.

Real-time analysis of G protein-coupled receptor reconstitution in a solubilized system.

Receptor based signaling mechanisms are the primary source of cellular regulation. The superfamily of G protein-coupled receptors is the largest and most ubiquitous of the receptor mediated processes. We describe here the analysis in real-time of the assembly and disassembly of soluble G protein-coupled receptor-G protein complexes. A fluorometric method was utilized to determine the dissociation of a fluorescent ligand from the receptor solubilized in detergent. The ligand dissociation rate differs between a receptor coupled to a G protein and the receptor alone. By observing the sensitivity of the dissociation of a fluorescent ligand to the presence of guanine nucleotide, we have shown a time- and concentration-dependent reconstitution of the N-formyl peptide receptor with endogenous G proteins. Furthermore, after the clearing of endogenous G proteins, purified Galpha subunits premixed with bovine brain Gbetagamma subunits were also able to reconstitute with the solubilized receptors. The solubilized N-formyl peptide receptor and Galpha(i3) protein interacted with an affinity of approximately 10(-6) m with other alpha subunits exhibiting lower affinities (Galpha(i3) > Galpha(i2) > Galpha(i1) Galpha(o)). The N-formyl peptide receptor-G protein interactions were inhibited by peptides corresponding to the Galpha(i) C-terminal regions, by Galpha(i) mAbs, and by a truncated form of arrestin-3. This system should prove useful for the analysis of the specificity of receptor-G protein interactions, as well as for the elucidation and characterization of receptor molecular assemblies and signal transduction complexes.

Internalization of the human N-formyl peptide and C5a chemoattractant receptors occurs via clathrin-independent mechanisms.

After stimulation by ligand, most G protein-coupled receptors (GPCRs) undergo rapid phosphorylation, followed by desensitization and internalization. In the case of the N-formyl peptide receptor (FPR), these latter two processing steps have been shown to be entirely dependent on phosphorylation of the receptor's carboxy terminus. We have previously demonstrated that FPR internalization can occur in the absence of receptor desensitization, indicating that FPR desensitization and internalization are regulated differentially. In this study, we have investigated whether human chemoattractant receptors internalize via clathrin-coated pits. Internalization of the FPR transiently expressed in HEK 293 cells was shown to be dependent upon receptor phosphorylation. Despite this, internalization of the FPR, as well as the C5a receptor, was demonstrated to be independent of the actions of arrestin, dynamin, and clathrin. In addition, we utilized fluorescence microscopy to visualize the FPR and beta(2)-adrenergic receptor as they internalized in the same cell, revealing distinct sites of internalization. Last, we found that a nonphosphorylatable mutant of the FPR, unable to internalize, was competent to activate p44/42 MAP kinase. Together, these results demonstrate not only that the FPR internalizes via an arrestin-, dynamin-, and clathrin-independent pathway but also that signal transduction to MAP kinases occurs in an internalization-independent manner.

Plug flow cytometry extends analytical capabilities in cell adhesion and receptor pharmacology.

BACKGROUND: Plug flow cytometry is a recently developed system for the automated delivery of multiple small boluses or "plugs" of cells or particles to the flow cytometer for analysis. Important system features are that sample plugs are of precisely defined volume and that the sample vessel need not be pressurized. We describe how these features enable direct cell concentration determinations and novel ways to integrate flow cytometers with other analytical instruments. METHODS: Adhesion assays employed human polymorphonuclear neutrophils (PMNs) loaded with Fura Red and Chinese hamster ovary (CHO) cells cotransfected with genes for green fluorescent protein (GFP) and human P-selectin. U937 cells expressing the human 7-transmembrane formyl peptide receptor were loaded with the fluorescent probe indo-1 for intracellular ionized calcium determinations. A computer-controlled syringe or peristaltic pump loaded the sample into a sample loop of the plug flow coupler, a reciprocating eight-port valve. When the valve position was switched, the plug of sample in the sample loop was transported to the flow cytometer by a pressure-driven fluid line. RESULTS: In stirred mixtures of PMNs and CHO cells, we used plug flow cytometry to directly quantify changes in concentrations of nonadherent singlet PMNs. This approach enabled accurate quantification of adherent PMNs in multicell aggregates. We constructed a novel plug flow interface between the flow cytometer and a cone-plate viscometer to enable real-time flow cytometric analysis of cell-cell adhesion under conditions of uniform shear. The High Throughput Pharmacology System (HTPS) is an instrument used for automated programming of complex pharmacological cell treatment protocols. It was interfaced via the plug flow coupling device to enable rapid (< 5 min) flow cytometric characterization of the intracellular calcium dose-response profile of U937 cells to formyl peptide. CONCLUSIONS: By facilitating the coupling of flow cytometers to other fluidics-based analytical instruments, plug flow cytometry has extended analytical capabilities in cell adhesion and pharmacological characterization of receptor-ligand interactions.

Analysis of free intracellular calcium by flow cytometry: multiparameter and pharmacologic applications.

Flow cytometry offers numerous advantages over traditional techniques for measuring intracellular Ca(2+) in lymphoid and nonlymphoid cells. In particular, the heterogeneity of cell responses can be defined by flow cytometry, and multiparameter analyses permit the determination of intracellular Ca(2+) in surface-marker-defined target cells as well as correlation of changes in Ca(2+) with other biochemical markers, including ligand binding. This article presents several established methods for measuring intracellular Ca(2+) by flow cytometry in lymphoid and nonlymphoid cells. Examples are provided for determination of Ca(2+) in human peripheral blood leukocytes and two human epithelial cell lines grown in monolayer. In addition, applications are reviewed or presented for correlating changes in intracellular Ca(2+) with other cell parameters, including cell cycle analysis, changes in cell membrane integrity, and the induction of apoptosis markers. Finally, a number of novel sample handling capabilities useful for performing kinetic analyses of Ca(2+) changes by flow cytometry are now available and one application is presented which is finding utility in pharmacologic studies.

Deficient homologous desensitization of formyl peptide receptors stably expressed in undifferentiated HL-60 cells.

The ability of formyl peptide receptors (FPRs) stably expressed in undifferentiated HL-60 cells to undergo ligand-induced desensitization was compared with their ability in normal and vector-transfected HL-60 cells following granulocyte differentiation with DMSO. fMet-Leu-Phe failed to induce uncoupling of FPRs from G-proteins in FPR-transfected cells, whereas uncoupling was induced in differentiated HL-60 cells and differentiated vector-transfected HL-60 cells, as determined by ligand-stimulated guanosine 5'-(gamma-thio)triphosphate (GTPgammaS) binding and GTPgammaS inhibition of fMet-Leu-Phe binding to isolated membranes. Immunoprecipitation of Galpha(i2) from solubilized, azidoanalide (AA-gammaGTP) photolabeled membranes showed that receptors in desensitized FPR-transfected HL-60 cells remained coupled to Galpha(i2), whereas desensitized receptors in differentiated HL-60 cell membranes were uncoupled from Galpha(i2). As determined by immunoblotting, Galpha(i2) expression was similar in undifferentiated and differentiated HL-60 cells and FPR-transfected cells. Ligand-stimulated receptor internalization and desensitization of calcium redistribution were similar in all three groups of cells. Immunoblotting also indicated that G-protein-coupled receptor kinases (GRKs) 2 and 4 were present in undifferentiated FPR-transfected HL-60 cells at 50% of the level seen in differentiated HL-60 cells. However, differentiation did not increase GRK2 or GRK4 expression, indicating that differences in GRK expression do not explain deficient desensitization. The data indicated that undifferentiated HL-60 cells are unable to induce homologous desensitization of FPRs.

Arrestin binding to the G protein-coupled N-formyl peptide receptor is regulated by the conserved "DRY" sequence.

Following activation by ligand, the N-formyl peptide receptor (FPR) undergoes processing events initiated by phosphorylation that lead to receptor desensitization and internalization. Our previous results have shown that FPR internalization can occur in the absence of receptor desensitization, suggesting that FPR desensitization and internalization are controlled by distinct mechanisms. More recently, we have provided evidence that internalization of the FPR occurs via a mechanism that is independent of the actions of arrestin, dynamin, and clathrin. In the present report, we demonstrate that stimulation of the FPR with agonist leads to a significant translocation of arrestin-2 from the cytosol to the membrane. Fluorescence microscopy revealed that the translocated arrestin-2 is highly colocalized with the ligand-bound FPR. A D71A mutant FPR, which does not undergo activation or phosphorylation in response to ligand, did not colocalize with arrestin-2. Surprisingly, an R123G mutant FPR, which does not bind G protein but does become phosphorylated and subsequently internalized, also did not bind arrestin. These results indicate that arrestin binding is not required for FPR internalization and demonstrate for the first time that a common motif, the conserved "DRY" domain of G protein-coupled receptors, is essential for phosphorylation-dependent arrestin binding, as well as G protein activation.

Differential phosphorylation paradigms dictate desensitization and internalization of the N-formyl peptide receptor.

Following activation by ligand, most G protein-coupled receptors undergo rapid phosphorylation. This is accompanied by a drastic decrease in the efficacy of continued or repeated stimulation, due to receptor uncoupling from G protein and receptor internalization. Such processing steps have been shown to be absolutely dependent on receptor phosphorylation in the case of the N-formyl peptide receptor (FPR). In this study, we report results that indicate that the mechanisms responsible for desensitization and internalization are distinct. Using site-directed mutagenesis of the serine and threonine residues of the FPR carboxyl terminus, we have characterized regions that differentially regulate these two processes. Whereas substitution of all 11 Ser/Thr residues in the carboxyl terminus prevents both desensitization and internalization, substitution of four Ser/Thr residues between 328-332 blocks desensitization but has no effect on internalization. Similarly, substitution of four Ser/Thr residues between positions 334 and 339 results in a deficit in desensitization but again no decrease in internalization, suggesting that phosphorylation at either site evokes receptor internalization, whereas maximal desensitization requires phosphorylation at both sites. These results also indicate that receptor internalization is not involved in the process of desensitization. Further analysis of the residues between 328-332 revealed that restoration either of Ser(328) and Thr(329) or of Thr(331) and Ser(332) was sufficient to restore desensitization, suggesting that phosphorylation within either of these two sites, in addition to sites between residues 334 and 339, is sufficient to produce desensitization. Taken together, these results indicate that the mechanisms involved in FPR processing (uncoupling from G proteins and internalization) are regulated differentially by phosphorylation at distinct sites within the carboxyl terminus of the FPR. The relevance of this paradigm to other G protein-coupled receptors is discussed.

The uncoupled state of the human formyl peptide receptor.

The formyl peptide receptor (FPR) has been widely used to study the kinetics of the interaction between ligand, receptor and G protein with real-time fluorescence methods. Because the wild type receptor rapidly signals, and is then desensitized and internalized once occupied by ligand, it has been difficult to study the uncoupled receptor form. We have examined a mutant form of the FPR expressed in U937 cells that does not bind G protein and is thus ideal to study the uncoupled form of the FPR in the intact cell. Using kinetic flow cytometry, we have measured the dissociation kinetics of a fluorescent ligand from this mutant in intact, permeabilized and fixed cells. We observed a novel uncoupled receptor form in the intact cell with a dramatically reduced off-rate (approximately 0.02 s-1) from LR in a broken cell preparation (approximately 0.2 s-1). Both receptor forms are retained in the presence of formaldehyde. We also observed this novel receptor form coexisting with the LRG complex when the wild type receptor is fixed in neutrophils or transfectants. These results complex when the wild type receptor is fixed in neutrophils o transfectants. These results lead us to suggest that there are distinct receptor structures in cells and membranes and that only a fraction of receptors in intact cells exist in the uncoupled form.

Multiple activation steps of the N-formyl peptide receptor.

The human N-formyl peptide receptor (FPR) is representative of a growing family of G protein-coupled receptors (GPCR) that respond to chemokines and chemoattractants. Despite the importance of this receptor class to immune function, relatively little is known about the molecular mechanisms involved in their activation. To reveal steps required for the activation of GPCR receptors, we utilized mutants of the FPR which have previously been shown to be incapable of binding and activating G proteins. For this study, the FPR mutants were expressed in human myeloid U937 cells and characterized for functions in addition to G protein coupling, such as receptor phosphorylation and ligand-induced receptor internalization. The results demonstrated that one of the mutants, R123G, though being unable to activate G protein, was capable of undergoing ligand-induced phosphorylation as well as internalization. Receptor internalization was monitored by following the fate of the ligand as well as by directly monitoring the fate of the receptor. The results with the R123G mutant were in contrast to those obtained for mutants D71A and R309G/E310A/R311G which, though being expressed at the cell surface and binding ligand, were incapable of being phosphorylated or internalized upon agonist stimulation. These results suggest that following ligand binding at least two "steps" are required for full activation of the wild-type FPR. That these observations may be of more general importance in GPCR-mediated signaling is suggested by the highly conserved nature of the mutants studied: D71, R123, and the site represented by amino acids 309-311 are very highly conserved throughout the entire superfamily of G protein-coupled receptors. Models of receptor activation based on the observed results are discussed.

Activation of mitogen-activated protein kinases by formyl peptide receptors is regulated by the cytoplasmic tail.

Wild type formyl peptide receptors (FPRwt) and receptors deleted of the carboxyl-terminal 45 amino acids (FPRdel) were stably expressed in undifferentiated HL-60 promyelocytes. Expression of FPRwt reconstituted N-formylmethionyl-leucyl-phenylalanine (FMLP)-stimulated extracellular signal-regulated kinase (ERK) and p38 kinase activity. Expression of FPRdel resulted in a 2-5-fold increase in basal ERK and p38 kinase activity, whereas FMLP failed to stimulate either mitogen-activated protein kinase (MAPK). Pertussis toxin abolished FMLP stimulation of both MAPKs in FPRwt cells but had no effect on either basal or FMLP-stimulated MAPK activity in FPRdel cells. FMLP stimulated a concentration-dependent increase in guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) binding in membranes from FPRwt but not FPRdel cells. GTPgammaS inhibited FMLP binding to FPRwt but not FPRdel membranes. Photoaffinity labeling with azidoanilide-[gamma-32P]GTP in the presence or absence of FMLP showed increased labeling only in FPRwt membranes. Immunoprecipitation of alphai2 and alphaq/11 from solubilized, photolabeled membranes showed that FPRwt were coupled to alphai2 but not to alphaq/11. FPRwt cells demonstrated calcium mobilization following stimulation with FMLP, whereas FPRdel cells showed no increase in intracellular calcium. We conclude that the carboxyl-terminal tail of FPRs is necessary for ligand-mediated activation of Gi proteins and MAPK cascades. Deletion of the carboxyl-terminal tail results in constitutive activation of ERK and p38 kinase through a Gi2-independent pathway.

Strategies for positioning fluorescent probes and crosslinkers on formyl peptide ligands.

Chemoattractant receptors represent a major subset of the G-protein coupled receptor (GPCR) family. One of the best characterized, the N-formyl peptide receptor (FPR), participates in host defense responses of neutrophils. The features of the ligand which regulate its interaction with the FPR are well-known. By manipulating these features we have developed new ligands to probe structural and mechanistic aspects of the peptide-receptor interaction. Three ligand groups have been developed: 1) ligands containing a Lys residue located in positions 2 through 7 that can be conjugated to FITC (N-formyl-Met1-Lys2-Phe3-Phe4, N-formyl-Met1-Leu2-Lys3-Phe4, N-formyl-Met1-Leu2-Phe3-Lys4, N-formyl-Met1-Leu2-Phe3-Phe4-Lys5, N-formyl-nLeu1-Leu2-Phe3-nLeu4-Tyr5-Lys6 and N-formyl-Met1-Leu2-Phe3-Phe4-Gly5-Gly6-Lys7; 2) fluorescent pentapeptide ligands (N-formyl-Met-X-Phe-Phe-Lys(FITC) where X = Leu, Ala, Val or Gly); and 3) small crosslinking ligands where the photoaffinity crosslinker 4-azidosalicylic acid (ASA) was conjugated to Lys in positions 3 and 4 and p-benzoyl-phenylalanine (Bpa) was located in position 2 in N-formyl-Met1-Bpa2-Phe3-Tyr4. The peptides were characterized according to activity and affinity in human neutrophils and cell lines transfected with FPR. All of the peptides were agonists, with parallel affinity and activity. In the first group, the peptide activity decreases as Lys is placed closer to the N-formyl group and the activity is improved by 1-3 orders of magnitude by conjugation with FITC. In the second group, the dissociation rate of the peptide from the receptor increases as position 2 is replaced by aliphatic amino acids with smaller alkyl groups. In the third group, crosslinking ligands remain biologically active, display nM affinity and covalently label the FPR.