Two active molecular phenotypes of the tachykinin NK1 receptor revealed by G-protein fusions and mutagenesis.

The NK1 neurokinin receptor presents two non-ideal binding phenomena, two-component binding curves for all agonists and significant differences between agonist affinity determined by homologous versus heterologous competition binding. Whole cell binding with fusion proteins constructed between either Galpha(s) or Galpha(q) and the NK1 receptor with a truncated tail, which secured non-promiscuous G-protein interaction, demonstrated monocomponent agonist binding closely corresponding to either of the two affinity states found in the wild-type receptor. High affinity binding of both substance P and neurokinin A was observed in the tail-truncated Galpha(s) fusion construct, whereas the lower affinity component was displayed by the tail-truncated Galpha(q) fusion. The elusive difference between the affinity determined in heterologous versus homologous binding assays for substance P and especially for neurokinin A was eliminated in the G-protein fusions. An NK1 receptor mutant with a single substitution at the extracellular end of TM-III-(F111S), which totally uncoupled the receptor from Galpha(s) signaling, showed binding properties that were monocomponent and otherwise very similar to those observed in the tail-truncated Galpha(q) fusion construct. Thus, the heterogenous pharmacological phenotype displayed by the NK1 receptor is a reflection of the occurrence of two active conformations or molecular phenotypes representing complexes with the Galpha(s) and Galpha(q) species, respectively. We propose that these molecular forms do not interchange readily, conceivably because of the occurrence of microdomains or "signal-transductosomes" within the cell membrane.

Disulfide bridge engineering in the tachykinin NK1 receptor.

As in most other seven-transmembrane receptors, the central disulfide bridge from the extracellular end of TM-III to the middle of the second extracellular loop was essential for ligand binding in the NK1 receptor. However, introduction of "extra", single Cys residues in the second extracellular loop, at positions where disease-associated Cys substitutions impair receptor function in the vasopressin V2 receptor and in rhodopsin, did not cause mispairing with the Cys residues involved in this central disulfide bridge. Cys residues were introduced in the N-terminal extension and in the third extracellular loop, respectively, in such a way that disulfide bridge formation could be monitored by loss of substance P binding and breakage of the bridge could be monitored by gain of ligand binding. This disulfide bridge formed spontaneously in the whole population of receptors and could be titrated with low concentrations of reducing agent, dithiothreitol. Another putative disulfide bridge "switch" was constructed at the extracellular ends of TM-V and -VI, i.e., at positions where a high-affinity zinc site previously had been constructed with His substitutions. Disulfide bridge formation at this position, monitored by loss of binding of the nonpeptide antagonist [3H]LY303.870, occurred spontaneously only in a small fraction of the receptors. It is concluded that disulfide bridges form readily between Cys residues introduced appropriately in the N-terminal extension and the third extracellular loop, whereas they form with more difficulty between Cys residues placed at the extracellular ends of the transmembrane segments even at positions where high-affinity metal ion sites can be constructed with His residues.

The type II IL-1 receptor interacts with the IL-1 receptor accessory protein: a novel mechanism of regulation of IL-1 responsiveness.

IL-1 binds to two types of receptors on the cell membrane, of which only type I (IL-1RI) transduces signals in concert with the coreceptor IL-1 receptor accessory protein (IL-1RAcP) while type II (IL-1RII) allegedly functions solely as ligand sink and decoy receptor without participating in IL-1 signaling. To investigate the regulatory role of IL-1RII on IL-1 responsiveness, a chimeric receptor encompassing the extracellular and transmembrane portions of IL-1RII and the cytoplasmic signal-transducing domain of IL-1RI was transfected into two murine EL-4-derived sublines that do or do not express IL-1RAcP, respectively. The chimeric receptor was able to transduce the IL-1 signal and induce IL-2 production only in the cell line which expressed IL-1RAcP, suggesting effective interaction between the extracellular domains of IL-1RII and IL-1RAcP in the presence of IL-1. The physical association of ligated IL-1RII with IL-1RAcP was proven by crosslinking experiments with radio-iodinated IL-1 and subsequent immunoprecipitations in normal human B cells and in EL-4 D6/76 cells transiently cotransfected with IL-1RII and IL-1RAcP, respectively. Based on these findings, it is proposed that upon IL-1 binding IL-1RII can recruit IL-1RAcP into a nonfunctional trimeric complex and thus modulate IL-1 signaling by subtracting the coreceptor molecule from the signaling IL-1RI. In this novel mechanism of coreceptor competition, the ratio between IL-1RII and IL-1RI becomes the central factor in determining the IL-1 responsiveness of a cell and the availability of IL-1RAcP becomes limiting for effective IL-1 signaling.

Human anaphylatoxin C4a is a potent agonist of the guinea pig but not the human C3a receptor.

The interaction of human anaphylatoxin C4a with the guinea pig (gp) and human (hu) C3a receptors (C3aR) was analyzed using human rC4a, which exhibited C4a-specific activity on guinea pig platelets. A gpC3aR of 475 residues with a large second extracellular loop and a peptide sequence approximately 60% identical to the huC3aR was isolated from a genomic DNA library and found to be expressed in guinea pig heart, lung, and spleen. HEK-293 cells cotransfected with this clone, and a cDNA encoding G alpha-16 specifically bound (Kd = 1.6+/-0.7 nM) and responded functionally to C3a with an intracellular calcium mobilization (ED50 = 0.18+/-0.02 nM). Human rC4a weakly bound to both the hu- and gpC3aR (IC50 > 1 microM). However, only HEK-293 cells expressing the gpC3aR responded functionally to rC4a (ED50 = 8.7+/-0.52 nM), while cells expressing the huC3aR did not (c < or = 1 microM). Thus, through an interaction with the C3aR, huC4a may elicit anaphylatoxic effects in guinea pigs but not in man.

The C terminus of the human C5a receptor (CD88) is required for normal ligand-dependent receptor internalization.

The biological effects of the potent inflammatory mediator C5a, a complement split product, on human neutrophils and monocytes are limited by the rapid internalization of its specific receptor (C5aR, CD88). The C terminus of the C5aR is phosphorylated after stimulation with C5a of phorbol ester, and this phosphorylation might lead to receptor internalization. In this context, we have studied the effects on C5aR internalization of C5a, phorbol 12-myristate 13-acetate (PMA), the protein kinase inhibitor staurosporine, and pertussis toxin on rat basophilic RBL.2H3 cells stably transfected with the human wild-type or mutant C5aR. C5aR mutants lacked either part of the cytosolic C terminus, including suggested major phosphorylation sites, or a putative phosphorylation motif for protein kinase C in the third cytosolic loop. Additionally, agonist-induced internalization was analyzed on HEK293 cells co-transfected with C5aR and the pertussis toxin-resistant G protein alpha subunit, G alpha 16. Staurosporine-sensitive agonist-dependent C5aR internalization could be detected, suggesting that C5aR phosphorylation, most likely of the C terminus, participates in this type of internalization. In contrast, PMA-induced C5aR internalization seems to be independent of putative phosphorylation sites in either the truncated section of the C terminus or the third cytosolic loop. The phorbol ester-induced C5aR internalization may, therefore, be caused by an indirect and less specific effect of protein kinase C on the internalization machinery. Manipulation of the pertussis toxin-sensitive or -resistant G protein-dependent signal transduction had no effect on ligand-induced internalization.

Expression cloning of the human C3a anaphylatoxin receptor (C3aR) from differentiated U-937 cells.

A cDNA clone encoding the human C3a anaphylatoxin receptor (C3aR) was isolated from a pcDNAI/Amp expression library prepared from U-937 cells which had been differentiated with dibutyryl cAMP to a macrophage-like phenotype. The cDNA clone contained an insert of 4.3 kbp and was able to confer to transfected human HEK-293 cells the capacity to bind specifically iodinated human C3a. Chinese hamster ovary cells co-transfected with this cDNA clone and a G-protein alpha subunit (G alpha-16) became functionally responsive to C3a and a C3a analog synthetic peptide, as measured by increased phosphoinositide hydrolysis. As inferred from the cDNA sequence, the clone encodes a 482-residue polypeptide with seven hydrophobic membrane-spanning helices and a high homology to the human C5a and formyl-Met-Leu-Phe receptors. Uniquely among the family of G-protein coupled receptors, the C3aR contains an exceptionally large second extracellular loop of approximately 175 residues. Northern hybridizations revealed an approximately 2.3-kb transcript as the major and an additional approximately 3.9 kb-transcript as a minor transcription product of the C3aR. The C3aR appears to be widely expressed in different lymphoid tissues, as shown by Northern hybridizations, providing evidence for a central role of the C3a anaphylatoxin in inflammatory processes.

Site-directed mutagenesis of conserved charged residues in the helical region of the human C5a receptor. Arg2O6 determines high-affinity binding sites of C5a receptor.

The human C5a receptor (C5aR) belongs to the family of G-protein-coupled receptors with seven transmembrane helices. This part of the molecule is thought to contain part of the ligand-binding pocket, specifically to bind the C-terminal Arg of human C5a. Guided by sequence similarity and molecular modelling studies, several residues including polar (Asn119, Thr168, Gln259) as well as all conserved charged amino acids in the upper transmembrane region of the C5aR (Asp37, Asp82, Arg175, Arg2O6, Asp282) were exchanged by site-directed mutagenesis. Receptor mutants were transiently expressed in COS cells and analyzed for altered binding behaviour and/or localization at the cell surface by immunofluorescence. For all residues, suitable mutants could be found that exhibited wild-type affinity towards the ligand, providing evidence against a major contribution of these residues to high-affinity ligand binding. Some mutants, however, exhibited a complete (Asp282-->Ala) or partial loss of ligand-binding capacity (Arg175-->Ala, Arg2O6-->Gln) despite adequate expression levels on the cell surface. This phenotype was further analyzed in the [Gln2O6]C5aR mutant: quantitative flow cytometric analysis of epitope-tagged receptor derivatives in 293 cells confirmed an equal level of wild-type and mutant C5aR on the cell surface. Competitive binding curves revealed the presence of only a small population (<10%) of high-affinity sites (Kd approximately 2 nM), which was functionally active at 20 nM in the heterologous Xenopus oocyte expression system after coexpression of G alpha-16. The number of high-affinity sites of wild-type and [Gln2O6]C5aR in 293 cells could be up-regulated by coexpression of Gi alpha-2 and down-regulated by GTP[gamma S]-mediated uncoupling of the G-protein receptor interaction in membrane preparations. These findings are compatible with a model in which the Arg2O6 residue located in the upper third of transmembrane helix V determines high-affinity binding in the human C5aR by affecting the intracellular G-protein coupling.

G alpha-16 complements the signal transduction cascade of chemotactic receptors for complement factor C5a (C5a-R) and N-formylated peptides (fMLF-R) in Xenopus laevis oocytes: G alpha-16 couples to chemotactic receptors in Xenopus oocytes.

The human leukocyte chemoattractant receptors for complement factor C5a (C5a-R) and N-formylated peptides (fMLF-R) are important members of the superfamily of G-protein coupled receptors (GPCR). Uniquely among the GPCR, these two receptors cannot be expressed in a functionally active form in the oocytes of the frog Xenopus laevis, but require substitution of total RNA of the myelomonocytic U-937 or HL-60 cell lines, respectively. Recently, it was reported that the C5a-R may couple to the alpha subunit of G-16. We have tested this G-protein for its ability to complement the signal transduction cascade of the C5a-R and fMLF-R in Xenopus oocytes. Injection of cRNA for the C5a-R in combination with G alpha-16 led to expression of a functional C5a-R as measured by ligand-induced whole cell current. In contrast to a previous report, the fMLF-R exhibited some residual functional activity when transiently expressed in Xenopus oocytes the extent of which could, however, substantially be increased by coexpression of G alpha-16. Thus, G alpha-16 complements the signal transduction cascade of both receptors in Xenopus laevis oocytes and is most likely the complementing factor present in the U-937 and HL-60 cell lines.