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Serpentine receptors relay hormonal or sensory stimuli to heterotrimeric guanine nucleotide-binding proteins (G proteins). In most G protein-coupled receptors (GPCRs), binding of the agonist ligand elicits both stimulation of the G protein and endocytosis of the receptor. We have begun to address whether these responses reflect the same sets of conformational changes in the receptor using constitutively active mutants of the human complement factor 5a receptor (C5aR). Two different mutant receptors both constitutively activate G protein-mediated responses, but one (F251A) is endocytosed only in response to ligand stimulation, while the other (NQ) is constitutively internalized in the absence of ligand. Both the constitutive and ligand-dependent endocytosis are accompanied by recruitment of beta-arrestin to the receptor. An inactivating mutation (N296A) complements the NQ mutation, producing a receptor that is activated only upon exposure to agonist; this revertant receptor (NQ/N296A) is nevertheless constitutively endocytosed. Thus one mutant (F251A) requires agonist for triggering endocytosis but not for activation of the downstream G protein signal, while another (NQ/N296A) behaves in the opposite fashion. Dissociation of two responses normally dependent on agonist binding indicates that the corresponding functions of an activated GPCR reflect different sets of changes in the receptor's conformation.
Serpentine receptors, a family of ligand-activated molecular switches, relay many different extracellular stimuli to heterotrimeric (αβγ) G proteins located on the cytoplasmic face of the plasma membrane. These receptors promote exchange of GDP for GTP bound to the α subunit of the heterotrimer, allowing the Gα-GTP and βγ subunits to separate and subsequently to activate intracellular effectors (1). Patterns of evolutionarily conserved amino acids distinguish six separate families of serpentine receptors (2), of which the rhodopsin-like family (several hundred members) is by far the largest (3). Mammalian serpentine receptors share with their orthologs in plants and yeast a conserved three-dimensional architecture with seven transmembrane α-helices arranged in a bundle (4). The activation mechanism of all these receptors must also be conserved, because mammalian receptors can activate yeast G proteins (5–7). The switch itself resides in the transmembrane helices: swapping extra- or intracellular loops between receptors changes specificity of ligand-binding or G protein-coupling, respectively, but leaves intact the capacity for ligand-dependent receptor activation, as summarized in (8).
Despite reports of the functional effects of an enormous number of mutations in many serpentine receptors, it has proved difficult to draw strong inferences about the conserved switch mechanism, in part because relatively few positions have been mutated in any one receptor (9). In addition, a receptor switch can exist in more than two positions, ‘off’ and ‘on’(10–13). The traditional view was that the active receptor conformation that stimulates one effector is identical to the conformation that stimulates a second effector or produces other agonist-dependent events, such as receptor endocytosis. This view is almost certainly an oversimplification; observations suggesting multiple active conformations have been reported from experiments with the beta-2 adrenergic receptor (14–16), the angiotensin II receptor (17), the N-formyl peptide receptor (18), and several chemokine receptors (18–22). Moreover, studies with the mu opioid receptor (MOR) have indicated that the MOR may have different ligand-selective activated conformations and that these conformations may be clinically relevant (23, 24).
In an attempt to identify G protein-coupled receptor (GPCR) residues that play key roles in the agonist-regulated switch, we subjected transmembrane helices of the receptor for complement factor 5a (the C5aR) to a comprehensive genetic analysis in yeast (25). This approach identified an essential cluster of evolutionarily conserved and functionally important residues in helices III, V, VI, and VII, located in the transmembrane core of the receptor and in close proximity to one another. Now a more detailed characterization of several mutations at the core positions, performed in the context of mammalian cells, shows that two different functions of an activated GPCR – agonist dependence of signaling to the downstream G protein and agonist dependence of endocytosis – can be completely dissociated. We therefore infer that these two signaling events depend on different sets of conformational changes in the GPCR.
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Some protein signaling machines work as simple off/on switches, but many can assume multiple different activated conformations, which allows them to generate more complex patterns of response to input stimuli. Examples include combinatorial integration of multiple stimuli [e.g. coincidence detectors like N-WASp (39) or NMDA-sensitive ion channels (40, 41)] and the qualitatively distinct output signals generated by entry of chemically different ligands into the binding pocket of a receptor [e.g. the estrogen receptor (42, 43)]. Our experiments with mutant C5aRs and observations of several other GPCRs (discussed further below) suggest that GPCRs belong in the last category: that is, GPCRs can assume multiple conformations to generate qualitatively different outputs.
In the absence of ligand, the WT C5aR and the three mutants present all four possible phenotypic combinations of constitutive signaling via G proteins and constitutive endocytosis: WT receptors show neither constitutive G protein signaling nor constitutive endocytosis (S–/E–), while the NQ, NQ/N296A combination, and F251A phenotypes are S+/E+, S–/E+, and S+/E–, respectively. The mutations that dissociate constitutive activation from constitutive endocytosis do not grossly affect receptor folding and transport to the plasma membrane, reduce affinities for binding C5a, or inhibit ability to signal to G proteins. Moreover, the mutants show maximal rates and extents of endocytosis (constitutive or in the presence of agonist, depending on the receptor) comparable to those seen with the agonist-bound WT receptor, and all recruit arrestin.
We imagine that the C5aR WT and mutant phenotypes represent four distinct molecular conformations. These conformations are determined by amino acid substitutions in the core of the helix bundle (Figure 1), distant (∼ 12–18 Å) from the receptor's cytoplasmic loops. These loops contact the trimeric G protein (8) and the endocytic machinery, including beta-arrestin (44), but the mutated residues do not. Thus the mutations – like agonist binding to a pocket oriented toward the extracellular fluid – effect conformational change indirectly, and at a distance. It is not clear how mutations at positions close to one another in the helix bundle produce such strikingly distinct functional phenotypes. It is worth noting, however, that amino acid residues cognate to F251, I124, L127, and N296 are highly conserved in other GPCRs of the rhodopsin family, and mutations at these positions often produce constitutive signaling activity (like NQ and F251A) or inactivate the receptor (like N296A) [(2,3) and references therein]. Conservation of primary structure and sensitivity to mutation in this region are consistent with the idea that this core region plays an important role in flipping the conformational switch, and/or modulating its output, in most GPCRs.
Our results and accumulating evidence from several laboratories suggest that the simple two-state (off/on) model of the GPCR switch is not correct. Experiments with opioid (23,24), beta-2 adrenergic (14–16), angiotensin II (17), and the N-formyl peptide (18) receptor have suggested that a single receptor can take on multiple distinct conformations. Strictly speaking, the C5aR mutant phenotypes do not by themselves indicate that the WT C5aR itself can take on multiple conformations in response to binding C5a; rather, each C5aR mutation may selectively promote a conformation that represents a subset of the overall conformation that normally accompanies activation by agonist. The fact that subtle alterations in the presumptive core switch of the C5aR produce different activated conformations does suggest, however, that different agonist ligands for a GPCR might flip the activation switch to produce multiple conformations.
Indeed, differential signaling upon stimulation by different ligands has been documented for several chemokine receptors, which, together with the N-formyl peptide receptor, are the closest relatives of the C5aR (45). Thus experiments with monoclonal antibodies raised against CC-chemokine receptor 5 that recognize different epitopes show profound differences in terms of receptor signaling, endocytosis, dimerization, competitive binding, and HIV-coreceptor function (22). The chemokines themselves are also able to provoke differing responses: CC-chemokine ligand 21 (CCL21) and CCL19 activate CCR7 signaling with comparable efficiency, but only CCL19 promotes internalization of CCR7 (21). Similar findings were reported for the actions of CCL5 and an N-terminal derivative thereof on another receptor, CCR1 (19,20). Arguments for two-state vs. multistate models of receptor activation have been thoroughly discussed (10–12).
Although our constitutively activating mutants provide useful experimental tools to dissect receptor activation from endocytosis, no naturally occurring, activating C5aR mutations have been reported. In other receptors, however, activating mutations can produce diseases. For example, mutations in the thyrotropin and luteotropin receptors cause thyrotoxicosis (46) and premature puberty (47), respectively. Curiously, the apparently activating R137H mutation in the V2 vasopressin receptor induces a loss-of-function phenotype, familial nephrogenic diabetes insipidus (48). In this case the mutant receptor – which shows constitutive signaling activity in vitro– undergoes constitutive arrestin-dependent desensitization and internalization. This V2 mutant phenotype thus resembles that produced by the NQ mutation in the C5aR, except that constitutive internalization of the V2 receptor in vivo removes so much receptor from the cell surface that signaling is abolished. In principle, mutations of serpentine receptors – like F251A in the C5aR – that activate G protein signaling without promoting constitutive regulation by arrestins and endocytosis could be even more dangerous physiologically, because their ‘on’ signal would not be counteracted by endocytosis and disappearance from the cell surface.