Site-directed mutagenesis of beta-adrenergic receptors. Identification of conserved cysteine residues that independently affect ligand binding and receptor activation

Using site-directed mutagenesis of the human beta 2-adrenergic receptor and continuous expression in B-82 cells, the role of 3 conserved cysteines in transmembrane domains and 2 conserved cysteines in the third extracellular domain in receptor function was examined. Cysteine was replaced with serine in each mutant receptor as this amino acid is similar to cysteine in size but it cannot form disulfide linkages. Replacement of cysteine residues 77 and 327, in the second and seventh transmembrane-spanning domains, respectively, had no effect on ligand binding or the ability of the receptor to mediate isoproterenol stimulation of adenylate cyclase. Substitution of cysteine 285, in the sixth transmembrane domain of the receptor, produced a mutant receptor with normal ligand-binding properties but a significantly attenuated ability to mediate stimulation of adenylate cyclase. Mutation of cysteine residues 190 and 191, in the third extracellular loop of the beta 2 receptor, had qualitatively similar effects on ligand binding and isoproterenol-mediated stimulation of adenylate cyclase. Replacement of either of these residues with serine produced mutant receptors that displayed a marked loss in affinity for both beta-adrenergic agonists and antagonists. Replacement of both cysteine 190 and 191 with serine had an even greater effect on the ability of the receptor to bind ligands. Consistent with the loss of Ser190 and/or Ser191 mutant receptor affinity for agonists was a corresponding shift to the right in the dose-response curve for isoproterenol-induced increases in intracellular cyclic AMP concentrations in cells expressing the mutant receptors. These data implicate one of the conserved transmembrane cysteine residues in the human beta 2-adrenergic receptor in receptor activation by agonists and also suggest that conserved cysteine residues in an extracellular domain of the receptor may be involved in ligand binding.     

TrkA immunoglobulin-like ligand binding domains inhibit spontaneous activation of the receptor

The extracellular region of the nerve growth factor (NGF) receptor, TrkA, contains two immunoglobulin (Ig)-like domains that are required for specific ligand binding. We have investigated the possible role of these two Ig-like domains in receptor dimerization and activation by using different mutants of the TrkA extracellular region. Deletions of each Ig-like domain, of both, and of the entire extracellular region were made. To probe the structural constraints on ligand-independent receptor dimerization, chimeric receptors were generated by swapping the Ig-like domains of the TrkA receptor for the third or fourth Ig-like domain of c-Kit. We also introduced single-amino-acid changes in conserved residues within the Ig-like domains of TrkA. Most of these TrkA variants did not bind NGF, and their expression in PC12nnr5 cells, which lack endogenous TrkA, promoted ligand-independent neurite outgrowth. Some TrkA mutant receptors induced malignant transformation of Rat-1 cells, as assessed by measuring proliferation in the absence of serum, anchorage-independent growth, and tumorigenesis in nude mice. These mutants exhibited constitutive phosphorylation and spontaneous dimerization consistent with their biological activities. Our data suggest that spontaneous dimerization of TrkA occurs when the structure of the Ig-like domains is altered, implying that the intact domains inhibit receptor dimerization in the absence of NGF.     

Down-regulation of mt1 melatonin receptors in rat ovary following estrogen exposure.

In this study, we have demonstrated that 2-[125I]-iodomelatonin binds specifically to rat ovarian granulosa cell (GC) membranes with high affinity (KD=83 pM; Bmax=3.28 fmol/mg protein). Using immunoblot analysis and an anti-mt1 melatonin receptor antibody, we have also detected mt1 melatonin receptors in rat ovary. Because melatonin has been reported to alter the steroidogenic responses of ovarian tissues to gonadotropins, a physiological role for intra-ovarian melatonin may exist. Thus, in order to investigate a possible intra-ovarian role for melatonin, we have used both an in vivo and in vitro model of follicular development. Treatment of immature (day 21) female rats with estradiol (E; 0.2 mg/d x 3 d; subcutaneous) was used to induce follicular growth. Membranes from both untreated (U) and E-treated animals' ovaries contained high-affinity 2-[125I]-iodomelatonin (I-MEL) binding sites (Kd=83 and 23 pM, respectively). Estradiol treatment in vivo caused a significant decrease (P<0.05) in binding of 2-[125I]-iodomelatonin to ovarian membranes with untreated animals' ovaries having a Bmax=3.28 fmol/mg protein vs. estradiol-treated animals' ovaries having a Bmax=0.92 fmol/mg protein. In addition, following Estradiol treatment, mt1 melatonin receptors in rat ovary were down-regulated (approximately 95%) using immunoblot analysis. Granulosa cells isolated from E-treated rats were further matured in vitro with testosterone (T) and the pituitary gonadotropin follicle-stimulating hormone (FSH). Granulosa cells were cultured with either T (10 ng/ml) or FSH (5.71 ng ovine FSH-20/ml) alone, or both FSH and T for 48 h. There was no statistically significant specific binding of 2-[125I]-iodomelatonin to GC membranes cultured with T or FSH alone. However, following a 48-h exposure to FSH and T in vitro specific 2-[125I]-iodomelatonin binding occurred with total 2-[125I]-iodomelatonin binding =3.15 [corrected] fmol/mg protein. Therefore, the existence of hormonally-regulated expression of high-affinity melatonin binding sites suggests that melatonin may have an important intra-ovarian physiological role.     

Photic regulation of mt(1) melatonin receptors and 2-iodomelatonin binding in the rat and Siberian hamster

We have investigated the photic regulation of melatonin receptors both at the level of binding capacity and mt(1) mRNA expression in the suprachiasmatic nucleus (SCN) and the pars tuberalis (PT) of the pituitary of two species: a highly photoperiodic one, the Siberian hamster, and a nonphotoperiodic one, the Wistar rat. This study has been performed by looking at the effect of a light pulse applied during the night on the two receptor parameters. The results show that the photic regulations of mt(1) mRNA expression and receptor density are distinct from each other in both the SCN and PT of the two species studied. They also show that, depending on the species and the structure, this regulation may implicate either the circadian clock or melatonin.     

Hemoglobin-binding site on human haptoglobin. Identification of lysyl residues participating in the binding.

The locus of human haptoglobin (Hp) molecules that interacts with human hemoglobin (Hb) was examined by the differential labeling technique. First, amino groups of Hp were extensively labeled with ethyl acetimidate (EAI) either in the free state (experiment A) or in the equimolar complex with Hb (experiment B). Each of the labeled Hp samples was reduced and S-carboxymethylated, and the remaining amino groups were then allowed to react with trinitrobenzenesulfonic acid (TNBS) under denatured conditions. Only 0.6 mol/mol of the trinitrophenyl (TNP) group was introduced into the heavy chain of Hp in experiment A, whereas 2.5 mol/mol of TNP groups were into the same chain in experiment B. The extent of TNP modification for the light chain was very low in either of the experiments. The amino acid residues carrying TNP groups in the heavy chain were identified by the peptide mapping procedure. They were Ile1, Lys136, and Lys218 in experiment B, and only Ile1 in experiment A. These findings suggest that epsilon-amino groups of Lys136 and Lys218 in the heavy chain are both situated within the Hb-binding locus of Hp, and that the alpha-amino group of Ile1 is buried inside the molecule either in the presence or absence of Hb.     

Chimeric Fc receptors identify ligand binding regions in human glycoprotein VI

Glycoprotein (GP) VI, a key receptor for collagen-induced platelet activation, recently emerged as a major target for developing new antithrombotics. However, little is known about its functional domains, which is a disadvantage for the rational development of antagonists. Our aim was to identify the structures determining GPVI specificity. GPVI presents homologies with members of the Ig superfamily (in particular with FcalphaRI) whose extracellular parts present two domains, D1 and D2 linked by a hinge interdomain. To identify the respective role of these domains in GPVI, we have substituted D1 and D2 by their FcalphaRI homologue in a soluble GPVI fusion protein (GPVI-Fc) and have modified the linker motif by mutagenesis. Proteins were tested for their binding to ligands and antibodies specific for GPVI and FcalphaRI. We demonstrate for the first time that D2 plays a specific and significant role in GPVI binding to collagen and that the hinge interdomain is critical for the binding to convulxin. Furthermore, binding to CRP requires elements of D1 and of the linker motif. Our results indicate that GPVI is unique amongst the receptors of its family as it uses different structural domains to interact with several agonists and provide evidence that different sites on GPVI constitute targets to develop antagonists of GPVI.     

Transmembrane domains 4 and 7 of the M(1) muscarinic acetylcholine receptor are critical for ligand binding and the receptor activation switch.

Activation of the muscarinic acetylcholine receptors requires agonist binding followed by a conformational change, but the ligand binding and conformation-switching residues have not been completely identified. Systematic alanine-scanning mutagenesis has been used to assess residues 142-164 in transmembrane helix 4 and 402-421 in transmembrane helix 7 of the M(1) muscarinic acetylcholine receptor. Several inward-facing amino acid side chains in the exofacial parts of transmembrane helices 4 and 7 contribute to acetylcholine binding. Alanine substitution of the aromatic residues in this group reduced signaling efficacy, suggesting that they may form part of a charge-stabilized aromatic cage, which triggers rotation and movement of the transmembrane helices. The mutation of adjacent residues modulated receptor activation, either reducing signaling or causing constitutive activation. In the buried endofacial section of transmembrane helix 7, alanine substitution mutants of the conserved NSXXNPXXY motif displayed strongly reduced signaling efficacy, despite having increased or unchanged acetylcholine affinity. These residues may have dual functions, forming intramolecular contacts that stabilize the receptor in the inactive ground state, but that are broken, allowing them to form new intramolecular bonds in the activated state. This conformational rearrangement is critical to produce a G protein binding site and may represent a key mechanism of receptor activation.     

Vasoactive intestinal polypeptide VPAC1 and VPAC2 receptor chimeras identify domains responsible for the specificity of ligand binding and activation.

In order to identify the receptor domains responsible for the VPAC1 selectivity of the VIP1 agonist, [Lys15, Arg16, Leu27] VIP (1-7)/GRF (8-27) and VIP1 antagonist, Ac His1 [D-Phe2, Lys15, Arg16, Leu27] VIP (3-7)/GRF (8-27), we evaluated their binding and functional properties on chimeric VPAC1/VPAC2 receptors. Our results suggest that the N-terminal extracellular domain is responsible for the selectivity of the VIP1 antagonist. Selective recognition of the VIP1 agonist was supported by a larger receptor area: in addition to the N-terminal domain, the first extracellular loop, as well as additional determinants in the distal part of the VPAC1 receptor were involved. Furthermore, these additional domains were critical for an efficient receptor activation, as replacement of EC1 in VPAC1 by its counter part in the VPAC2 receptor markedly reduced the maximal response.     

Identification of ligand binding determinants of the prolactin receptor.

The prolactin (PRL) receptor, a lactogen- and primate somatogen-binding protein, is a member of an expanding superfamily (cytokine/growth hormone (GH)/PRL) of single membrane-spanning receptors. Two features commonly shared among this group of proteins are the presence of two pairs of cysteines, generally found in the N-terminal region of the extracellular domain, and a WSxWS (WS) motif, frequently located proximal to the transmembrane domain. We have recently shown the 4 cysteines to be critical to the maintenance of the structural and functional integrity of the PRL-receptor. In the present study, we prepared a set of eight chimeric rat PRL/human GH receptors and several alanine mutants, to assess the importance of the Cys-rich domain (residues 12-68) in confering specificity to PRL binding. The role of the WS motif in high affinity binding was also investigated. Binding of 125I-labeled ovine PRL or human GH to membrane preparations from COS-7 cells transiently expressing the mutant receptors have defined a region within the first disulfide loop (residues Arg13, Asp16, Glu18) and the set of lactogen-specific sequences between the two pairs of cysteines as key determinants of PRL-binding specificity, which converge to form a patch on a two-dimensional model of the PRL receptor. We also demonstrate that, although PRL- and GH-specific determinants overlap in certain areas, they are not identical. Finally, substitution of the WS motif with alanine residues precludes high affinity binding to ovine PRL and human GH and suggests that this structural element may provide a target site for the interaction of an accessory protein necessary for the formation of a high-affinity receptor complex.     

Chimeric Fc receptors identify functional domains of the murine high affinity receptor for IgG.

Chimeric Fc gamma R have been generated between the mouse high affinity receptor for IgG (Fc gamma RI) and the low affinity receptor for IgG (Fc gamma RII) by exchanging the first two domains of the three-domain extracellular structure of Fc gamma RI with the homologous two-domain extracellular structure of Fc gamma RII. Studies of the affinity and specificity of binding of mouse Ig classes to these receptors defined functional regions of Fc gamma RI and showed some surprising results. After removal of the third extracellular domain of Fc gamma RI, the remaining two domains (domains 1 and 2) retained the capacity to bind Ig in the form of immune complexes, however, they bound monomeric IgG2a with a reduced affinity. Surprisingly, these two domains in the absence of the third domain bound not only IgG2a but also IgG1 and IgG2b, i.e., the third domain of Fc gamma RI suppresses the intrinsic capacity of the first two domains to act as a low affinity Fc gamma RII-like molecule. Linking the third extracellular domain of Fc gamma RI to the two extracellular domains of Fc gamma RII resulted in a receptor that retained the specificity and affinity of Fc gamma RII. Thus, the removal of domain 3 from Fc gamma RI resulted in the conversion of Fc gamma RI to an "Fc gamma RII-like" receptor. These findings indicate that domains 1 and 2 of Fc gamma RI form an Ig-binding motif, and although domain 3 is not essential for Fc binding by Fc gamma RI, it plays a crucial role in determining the specific high affinity interaction of Fc gamma RI with IgG2a.     

Role of charged residues in coupling ligand binding and channel activation in the extracellular domain of the glycine receptor

The glycine receptor is a member of the ligand-gated ion channel receptor superfamily that mediates fast synaptic transmission in the brainstem and spinal cord. Following ligand binding, the receptor undergoes a conformational change that is conveyed to the transmembrane regions of the receptor resulting in the opening of the channel pore. Using the acetylcholine-binding protein structure as a template, we modeled the extracellular domain of the glycine receptor alpha1-subunit and identified the location of charged residues within loops 2 and 7 (the conserved Cys-loop). These loops have been postulated to interact with the M2-M3 linker region between the transmembrane domains 2 and 3 as part of the receptor activation mechanism. Charged residues were substituted with cysteine, resulting in a shift in the concentration-response curves to the right in each case. Covalent modification with 2-(trimethylammonium) ethyl methanethiosulfonate was demonstrated only for K143C, which was more accessible in the open state than the closed state, and resulted in a shift in the EC50 toward wild-type values. Charge reversal mutations (E53K, D57K, and D148K) also impaired channel activation, as inferred from increases in EC50 values and the conversion of taurine from an agonist to an antagonist in E53K and D57K. Thus, each of the residues Glu-53, Asp-57, Lys-143, and Asp-148 are implicated in channel gating. However, the double reverse charge mutations E53K:K276E, D57K:K276E, and D148K:K276E did not restore glycine receptor function. These results indicate that loops 2 and 7 in the extracellular domain play an important role in the mechanism of activation of the glycine receptor although not by a direct electrostatic mechanism.     

Characterization of insulin/IGF hybrid receptors: contributions of the insulin receptor L2 and Fn1 domains and the alternatively spliced exon 11 sequence to ligand binding and receptor activation

The IR (insulin receptor) and IGFR (type I insulin-like growth factor receptor) are found as homodimers, but the respective pro-receptors can also heterodimerize to form insulin-IGF hybrid receptors. There are conflicting data on the ligand affinity of hybrids, and especially on the influence of different IR isoforms. To investigate further the contribution of individual ligand binding epitopes to affinity and specificity in the IR/IGFR family, we generated hybrids incorporating both IR isoforms (A and B) and IR/IGFR domain-swap chimaeras, by ectopic co-expression of receptor constructs in Chinese hamster ovary cells, and studied ligand binding using both radioligand competition and bioluminescence resonance energy transfer assays. We found that IR-A-IGFR and IR-B-IGFR hybrids bound insulin with similar relatively low affinity, which was intermediate between that of homodimeric IR and homodimeric IGFR. However, both IR-A-IGFR and IR-B-IGFR hybrids bound IGF-I and IGF-II with high affinity, at a level comparable with homodimeric IGFR. Incorporation of a significant fraction of either IR-A or IR-B into hybrids resulted in abrogation of insulin- but not IGF-I-stimulated autophosphorylation. We conclude that the sequence of 12 amino acids encoded by exon 11 of the IR gene has little or no effect on ligand binding and activation of IR-IGFR hybrids, and that hybrid receptors bind IGFs but not insulin at physiological concentrations regardless of the IR isoform they contained. To reconstitute high affinity insulin binding within a hybrid receptor, chimaeras in which the IGFR L1 or L2 domains had been replaced by equivalent IR domains were co-expressed with full-length IR-A or IR-B. In the context of an IR-A-IGFR hybrid, replacement of IR residues 325-524 (containing the L2 domain and part of the first fibronectin domain) with the corresponding IGFR sequence increased the affinity for insulin by 20-fold. We conclude that the L2 and/or first fibronectin domains of IR contribute in trans with the L1 domain to create a high affinity insulin-binding site within a dimeric receptor.     

Mapping ligand binding domains in chimeric fibroblast growth factor receptor molecules. Multiple regions determine ligand binding specificity

Fibroblast growth factors (FGFs) mediate essential cellular functions by activating one of four alternatively spliced FGF receptors (FGFRs). To determine the mechanism regulating ligand binding affinity and specificity, soluble FGFR1 and FGFR3 binding domains were compared for activity. FGFR1 bound well to FGF2 but poorly to FGF8 and FGF9. In contrast, FGFR3 bound well to FGF8 and FGF9 but poorly to FGF2. The differential ligand binding specificity of these two receptors was exploited to map specific ligand binding regions in mutant and chimeric receptor molecules. Deletion of immunoglobulin-like (Ig) domain I did not effect ligand binding, thus localizing the binding region(s) to the distal two Ig domains. Mapping studies identified two regions that contribute to FGF binding. Additionally, FGF2 binding showed positive cooperativity, suggesting the presence of two binding sites on a single FGFR or two interacting sites on an FGFR dimer. Analysis of FGF8 and FGF9 binding to chimeric receptors showed that a broad region spanning Ig domain II and sequences further N-terminal determines binding specificity for these ligands. These data demonstrate that multiple regions of the FGFR regulate ligand binding specificity and that these regions are distinct with respect to different members of the FGF family.     

Identification of the hydrophobic ligand binding pocket of the S1P1 receptor

Sphingosine 1-phosphate (S1P), a naturally occurring sphingolipid mediator and also a second messenger with growth factor-like actions in almost every cell type, is an endogenous ligand of five G protein-coupled receptors (GPCRs) in the endothelial differentiation gene family. The lack of GPCR crystal structures sets serious limitations to rational drug design and in silico searches for subtype-selective ligands. Here we report on the experimental validation of a computational model of the ligand binding pocket of the S1P1 GPCR surrounding the aliphatic portion of S1P. The extensive mutagenesis-based validation confirmed 18 residues lining the hydrophobic ligand binding pocket, which, combined with the previously validated three head group-interacting residues, now complete the mapping of the S1P ligand recognition site. We identified six mutants (L3.43G/L3.44G, L3.43E/L3.44E, L5.52A, F5.48G, V6.40L, and F6.44G) that maintained wild type [32P]S1P binding with abolished ligand-dependent activation by S1P. These data suggest a role for these amino acids in the conformational transition of S1P1 to its activated state. Three aromatic mutations (F5.48Y, F6.44G, and W6.48A) result in differential activation, by S1P or SEW2871, indicating that structural differences between the two agonists can partially compensate for differences in the amino acid side chain. The now validated ligand binding pocket provided us with a pharmacophore model, which was used for in silico screening of the NCI, National Institutes of Health, Developmental Therapeutics chemical library, leading to the identification of two novel nonlipid agonists of S1P1.     

Amino acid residues conferring ligand binding properties of prostaglandin I and prostaglandin D receptors. Identification by site-directed mutagenesis

Using chimeras of the mouse prostaglandin (PG) I receptor (mIP) and the mouse PGD receptor (mDP), we previously revealed that the cyclopentane ring recognition by these receptors is specified by a region from the first to third transmembrane domain of each receptor; recognition by this region of mIP is broad, accommodating the D, E, and I types of cyclopentane rings, whereas that of mDP binds the D type of PGs alone (Kobayashi, T., Kiriyama, M., Hirata, T., Hirata, M., Ushikubi, F., and Narumiya, S. (1997) J. Biol. Chem. 272, 15154-15160). In the present study, we performed a more detailed chimera analysis, and narrowed the domain for the ring recognition to a region from the first transmembrane domain to the first extracellular loop. One chimera with the replacement of the second transmembrane domain and the first extracellular loop of mDP with that of mIP bound only iloprost. The amino acid substitutions in this chimera suggest that Ser(50) in the first transmembrane domain of mIP confers the broad ligand recognition of mIP and that Lys(75) and Leu(83) in the second transmembrane domain of mDP confer the high affinity to PGD(2) and the strict specificity of ligand binding of mDP, respectively.     

Assembly and ligand binding properties of the water-soluble extracellular domains of the glutamate receptor 1 subunit

High resolution structural studies of models of glutamate receptors (GluRs) have been limited to monomeric models of the ligand-binding site. To obtain oligomeric models of glutamate receptors that can reveal more complete structural information, we examined the assembly and ligand binding properties of two truncated versions of the GluR1 subunit. The first version, GluR1-WS, consisted of only the N-terminal extracellular segment (Ala(1)-Glu(520)) bridged by a synthetic linker to the second extracellular domain (Asn(615)-Gly(790)). The second version, GluR1-M1, consisted of the first N-terminal extracellular domain (Ala(1)-Glu(520)) bridged by a synthetic linker to a second segment containing the second extracellular domain, the third transmembrane domain, and the intracellular C-terminal domain (Asn(615)-Leu(889)). When expressed in Xenopus oocytes, GluR-WS was secreted and water-soluble; GluR1-M1 was displayed on the surface of oocytes. GluR1-WS exhibited a velocity sedimentation profile that was consistent with assembly of homooligomers and bound the glutamate receptor agonist alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid with high affinity. These findings show that the extracellular domains of GluR1 that are sufficient for ligand binding apparently are sufficient for subunit assembly and might be a suitable target for structural studies of a water-soluble GluR1 oligomer.     

The mt1 melatonin receptor and RORbeta receptor are co-localized in specific TSH-immunoreactive cells in the pars tuberalis of the rat pituitary.

The pars tuberalis (PT) of the pituitary represents an important target site for the time-pacing pineal hormone melatonin because it expresses a large number of mt1 receptors. Functional studies suggest that the PT mediates the seasonal effects of melatonin on prolactin (PRL) secretion. The aim of this study was the characterization of the phenotype of melatonin-responsive cells. Furthermore, we determined whether RORbeta, a retinoid orphan receptor present in the PT, was co-expressed in the same cells. We combined nonradioactive in situ hybridization (ISH) with hapten-labeled riboprobes for detection of the receptors and immunocytochemistry (ICC) for detection of alphaGSU (alpha-glycoprotein subunit), betaTSH, betaFSH, betaLH, GH, PRL, and ACTH. Expression of mt1 mRNA was found in small round cells, co-localized with alphaGSU and betaTSH. However, not all betaTSH-containing cells expressed mt1 mRNA. The distribution of mt1- and RORbeta-positive cells appeared to overlap, although more cells were labeled for RORbeta than for mt1. Gonadotrophs, as well as other pars distalis cell types, were never labeled for mt1 melatonin receptor. Therefore, this study identifies the "specific" cells of the PT as the mt1 melatonin receptor-expressing cells.