Expression and characterization of the naturally occurring mutation L394R in human gamma-glutamyl carboxylase

Patients with mutation L394R in gamma-glutamyl carboxylase have a severe bleeding disorder because of decreased biological activities of all vitamin K-dependent coagulation proteins. Vitamin K administration partially corrects this deficiency. To characterize L394R, we purified recombinant mutant L394R and wild-type carboxylase expressed in baculovirus-infected insect cells. By kinetic studies, we analyzed the catalytic activity of mutant L394R and its binding to factor IX's propeptide and vitamin KH(2). Mutant L394R differs from its wild-type counterpart as follows: 1) 110-fold higher K(i) for Boc-mEEV, an active site-specific, competitive inhibitor of FLEEL; 2) 30-fold lower V(max)/K(m) toward the substrate FLEEL in the presence of the propeptide; 3) severely reduced activity toward FLEEL carboxylation in the absence of the propeptide; 4) 7-fold decreased affinity for the propeptide; 5) 9-fold higher K(m) for FIXproGla, a substrate containing the propeptide and the Gla domain of human factor IX; and 6) 5-fold higher K(m) for vitamin KH(2). The primary defect in mutant L394R appears to be in its glutamate-binding site. To a lesser degree, the propeptide and KH(2) binding properties are altered in the L394R mutant. Compared with its wild-type counterpart, the L394R mutant shows an augmented activation of FLEEL carboxylation by the propeptide.     

A constitutively active mutant of the human lutropin receptor (hLHR-L457R) escapes lysosomal targeting and degradation

Using biochemical and imaging approaches, we examined the postendocytotic fate of the complex formed by human choriogonadotropin (hCG) and a constitutively active mutant of the human lutropin receptor (hLHR-L457R) found in a boy with precocious puberty and Leydig cell hyperplasia. After internalization, some of the complex formed by the hLHR-wild type (hLHR-wt) and hCG recycles to the cell surface, and some is found in lysosomes where the hormone is degraded. In contrast, the complex formed by the hLHR-L457R and hCG is not routed to the lysosomes, most of it is recycled to the cell surface and hormone degradation is barely detectable. For both, hLHR-wt and -L457R, there is an hCG-induced loss of cell surface receptors that accompanies internalization but this loss cannot be prevented by leupeptin. The removal of recycling motifs of the hLHR by truncation of the C-terminal tail at residue 682 greatly enhances the lysosomal accumulation of the hormone-receptor complexes formed by the hLHR-wt or the L457R mutant, the degradation of the internalized hormone, and the loss of cell surface receptors. The degradation of the hormone internalized by these mutants as well as the loss of cell surface receptors is largely prevented by leupeptin. These results highlight a previously unrecognized complexity in the postendocytotic trafficking of the hLHR and document a clear difference between the properties of the constitutively active mutant and the agonist-activated hLHR-wt. This lack of lysosomal degradation of the L457R mutant could contribute to its constitutive activity by prolonging the duration of signaling.     

Pleiotropic effects of substitutions of a highly conserved leucine in transmembrane helix III of the human lutropin/choriogonadotropin receptor with respect to constitutive activation and hormone responsiveness

It has been shown previously that a naturally occurring mutation of the human LH/CG receptor (hLHR), which replaces L457 in helix III with arginine, results in a receptor that constitutively elevates basal cAMP but does not respond to human CG (hCG) with further cAMP production. In the present study, substitutions of L457 with several amino acids were examined. The constitutive activation of cAMP production was observed only when L457 was replaced with a positively charged residue. Although constitutive activation of the inositol phosphate pathway could not be detected when measuring inositol phosphate production, the use of a more sensitive reporter gene assay for protein kinase C activation revealed the constitutive activation of this pathway by the R- and K-substituted mutants. Therefore, L457 of the hLHR plays a key role in stabilizing the receptor in an inactive conformation. Molecular modeling shows that the insertion of R, K, or H at position 457 triggers the receptor transition toward an active state due to the proximity of an anionic amino acid, D578, in helix VI. These substitutions cause perturbations in helix III-helix VI and helix III-helix VII interactions that culminate in the opening of a solvent-accessible site in the cytosolic domains potentially involved in Gs recognition. Interestingly, L457R was completely unresponsive and the K- and H-substituted L457 hLHR mutants were significantly blunted in their cAMP responses to hCG stimulation. Cells expressing L457R were also unresponsive to hCG with regards to increased inositol phosphate production. Other substitutions of L457 were identified, though, that selectively permit the hormonal stimulation of only one of the two signaling pathways. These results suggest a pivotal role for L457 in hormone-stimulated signal transduction by the hLHR.     

A naturally occurring mutation of the opsin gene (T4R) in dogs affects glycosylation and stability of the G protein-coupled receptor

Rho (rhodopsin; opsin plus 11-cis-retinal) is a prototypical G protein-coupled receptor responsible for the capture of a photon in retinal photoreceptor cells. A large number of mutations in the opsin gene associated with autosomal dominant retinitis pigmentosa have been identified. The naturally occurring T4R opsin mutation in the English mastiff dog leads to a progressive retinal degeneration that closely resembles human retinitis pigmentosa caused by the T4K mutation in the opsin gene. Using genetic approaches and biochemical assays, we explored the properties of the T4R mutant protein. Employing immunoaffinity-purified Rho from affected RHO(T4R/T4R) dog retina, we found that the mutation abolished glycosylation at Asn(2), whereas glycosylation at Asn(15) was unaffected, and the mutant opsin localized normally to the rod outer segments. Moreover, we found that T4R Rho(*) lost its chromophore faster as measured by the decay of meta-rhodopsin II and that it was less resistant to heat denaturation. Detergent-solubilized T4R opsin regenerated poorly and interacted abnormally with the G protein transducin (G(t)). Structurally, the mutation affected mainly the "plug" at the intradiscal (extracellular) side of Rho, which is possibly responsible for protecting the chromophore from the access of bulk water. The T4R mutation may represent a novel molecular mechanism of degeneration where the unliganded form of the mutant opsin exerts a detrimental effect by losing its structural integrity.     

A urokinase-sensitive region of the human urokinase receptor is responsible for its chemotactic activity.

The role of urokinase-type plasminogen activator (uPA) and its receptor (uPAR/CD87) in cell migration and invasion is well substantiated. Recently, uPA has been shown to be essential in cell migration, since uPA-/- mice are greatly impaired in inflammatory cell recruitment. We have shown previously that the uPA-induced chemotaxis requires interaction with and modification of uPAR/CD87, which is the true chemoattracting molecule acting through an unidentified cell surface component which mediates this cell surface chemokine activity. By expressing and testing several uPAR/CD87 variants, we have located and functionally characterized a potent uPAR/CD87 epitope that mimics the effects of the uPA-uPAR interaction. The chemotactic activity lies in the region linking domains 1 and 2, the only protease-sensitive region of uPAR/CD87, efficiently cleaved by uPA at physiological concentrations. Synthetic peptides carrying this epitope promote chemotaxis and activate p56/p59(hck) tyrosine kinase. Both chemotaxis and kinase activation are pertussis toxin sensitive, involving a Gi/o protein in the pathway.     

Charged residues at the intracellular boundary of transmembrane helices 2 and 3 independently affect constitutive activity of Kaposi's sarcoma-associated herpesvirus G protein-coupled receptor

Because charged residues at the intracellular ends of transmembrane helix (TMH) 2 and TMH3 of G protein-coupled receptors (GPCRs) affect signaling, we performed mutational analysis of these residues in the constitutively signaling Kaposi's sarcoma-associated herpesvirus GPCR (KSHV-GPCR). KSHV-GPCR contains the amino acid sequence Val-Arg-Tyr rather than the Asp/Glu-Arg-Tyr ((D/E)RY) motif at the intracellular end of TMH3. Mutation of Arg-143 to Ala (R143A) or Gln (R143Q) abolished constitutive signaling whereas R143K exhibited 50% of the basal activity of KSHV-GPCR. R143A was not stimulated by agonist, whereas R143Q was stimulated by growth-related oncogene-alpha, and R143K, similar to KSHV-GPCR, was stimulated further. These findings show that Arg-143 is critical for signal generation in KSHV-GPCR. In other GPCRs, Arg in this position may act as a signaling switch by movement of its sidechain from a hydrophilic pocket in the TMH bundle to a position outside the bundle. In rhodopsin, the Arg of Glu-Arg-Tyr interacts with the adjacent Asp to constrain Arg outside the TMH bundle. V142D was 70% more active than KSHV-GPCR, suggesting that an Arg residue, which is constrained outside the bundle by interacting with Asp-142, leads to a receptor that signals more actively. Because the usually conserved Asp in the middle of TMH2 is not present in KSHV-GPCR, we tested whether Asp-83 at the intracellular end of TMH2 was involved in signaling. D83N and D83A were 110 and 190% more active than KSHV-GPCR, respectively. The double mutant D83A/V142D was 510% more active than KSHV-GPCR. That is, cosubstitutions of Asp-83 by Ala and Val-142 by Asp act synergistically to increase basal signaling. A model of KSHV-GPCR predicts that Arg-143 interacts with residues in the TMH bundle and that the sidechain of Asp-83 does not interact with Arg-143. These data are consistent with the hypothesis that Arg-143 and Asp-83 independently affect the signaling activity of KSHV-GPCR.     

Contribution of a putative salt bridge and backbone dynamics in the structural instability of human prion protein upon R208H mutation

Molecular dynamics simulation method is used to assess the contribution of a disease-associated salt bridge in the early stages of the conformational rearrangement of human prion protein upon Arg²⁰⁸→His mutation, which causes Creutzfeldt-Jakob disease. Previous investigations have suggested that the breakage of this putative salt bridge (D¹⁴⁴/E¹⁴⁶ ↔ Arg²⁰⁸) between helix 1 and helix 3 is responsible for such a mutation-driven process. So far, no experimental data has been reported in order to distinguish the contribution of this single salt bridge in the initial steps of amyloid formation. Consequently, we decided to investigate the role of this salt bridge in early conformational rearrangements. To remove the salt bridge without perturbations in the backbone structure, the neutralized states of the involved residues were used. Three 10-ns molecular dynamics simulations on three initial structures have been performed. The results revealed that the early stages of the conformational rearrangements, against common belief, are mainly associated with the mutation-induced global changes in the backbone dynamics but not with the breaking of the salt bridge.     

The local environment at the cytoplasmic end of TM6 of the mu opioid receptor differs from those of rhodopsin and monoamine receptors: introduction of an ionic lock between the cytoplasmic ends of helices 3 and 6 by a L6.30(275)E mutation inactivates the mu opioid receptor and reduces the constitutive activity of its T6.34(279)K mutant

Activation of rhodopsin and monoamine G protein-coupled receptors (GPCRs) has been proposed to involve in part the disruption of a conserved E6.30-R3.50 ionic interaction between transmembrane segments (TMs) 3 and 6. However, this interaction does not occur in the opioid receptors, which have L275 at 6.30. On the basis of our findings that mutations of T6.34(279) to K and D produced, respectively, a constitutively active and an inactive form of the mu opioid receptor, we previously suggested that the functional role of the 6.30(275) residue could be assumed by T6.34(279), but the interplay between residues at positions 6.30 and 6.34 remained unresolved. In this study, we examined the effects of introducing an E in position 6.30(275) of the wild type (WT) and of the T6.34(279) mutants of the mu opioid receptor to compare the participation of the 6.30 locus in molecular events during activation in this receptor with its role in other GPCRs. The L6.30(275)E and the L6.30(275)E/T6.34(279)D mutants displayed no constitutive activity and could not be activated by the agonist DAMGO or morphine. The L6.30(275)E/T6.34(279)K mutant had some constitutive activity, but much less than the T6.34(279)K mutant, and could be activated by both agonists. The rank order of affinity for the agonist DAMGO is as follows: T6.34(279)K > WT congruent with L6.30(275)E/T6.34(279)K > L6.30(275)E congruent with T6.34(279)D > L6.30(275)E/T6.34(279)D; however, all constructs have a similar affinity for the antagonist [(3)H]diprenorphine. These data are interpreted in the context of interactions with the conserved R3.50(165) in TM3. When L6.30(275) is mutated to E, the favorable E6.30(275)-R3.50(165) interaction stabilizes an inactive state, as in rhodopsin, and hence reduces the activities of T6.34(279) mutants. Thus, the mu opioid receptor is shown to be different from rhodopsin and monoamine GPCRs, of which the WTs with native E6.30 can be activated, and the 6.34D or 6.34K mutants display enhanced constitutive activities. Our molecular modeling results suggest that some specific differences in local geometry at the cytoplasmic ends of TM5 and TM6 may account in part for the observed differences in the molecular mechanisms of receptor activation.     

SH3 domain of the phosphatidylinositol 3-kinase regulatory subunit is responsible for the formation of a sequestration complex with insulin receptor substrate-1

Class IA phosphatidylinositol 3-kinase (PI 3-kinase), which is composed of a 110 kDa catalytic subunit and a regulatory subunit, plays a key role in most insulin dependent cellular responses. To date, five mammalian regulatory subunit isoforms have been identified, including two 85 kDa proteins (p85α and p85β), two 55 kDa proteins (p55γ and p55α), and one 50 kDa protein (p50α). In the present study, we overexpressed these recombinant proteins, tagged with green fluorescent proteins (GFP), in CHO-IR cells and investigated intracellular localizations in both the presence and the absence of insulin stimulation. Interestingly, in response to insulin, only p85α and p85β redistributed to isolated foci in the cells, while both were present throughout the cytoplasm in quiescent cells. In contrast, p55s accumulated in the perinuclear region irrespective of insulin stimulation, while p50α behaved similarly to control GFP. Immunofluorescent antibodies against endogenous IRS-1 revealed IRS-1 to be co-localized in the p85 foci in response to insulin. As both insulin receptors and p110α catalytic subunits were absent from these foci on immunofluorescence study, only p85 and IRS-1 were suggested to form a sequestration complex in response to insulin. To determine the domain responsible for IRS-1 complex formation, we prepared and overexpressed the SH3 domain deletion mutant of p85α in CHO-IR cells. This mutant failed to form foci, suggesting the SH3 domain of regulatory subunits to be responsible for formation of the p85-IRS-1 sequestration complex. In conclusion, our study revealed the SH3 domain of PI 3-kinase to play a critical role in intracellular localizations, including formation of foci with IRS-1 in response to insulin.     

Functional characterization of the new human GABA(A) receptor mutation beta3(R192H)

We screened 124 individuals for single nucleotide polymorphisms of the alpha1, beta3 and gamma2 genes of the GABA(A) receptor in the regions corresponding to the ligand-binding domains on the protein level. In a patient with chronic insomnia, a missense mutation was found in the gene of the beta3 subunit. This mutation results in the substitution of the amino acid residue arginine for histidine in position 192 (beta3(R192H)). The patient was found to be heterozygous for this mutation. Functional analysis of human alpha1beta3(R192H)gamma2S GABA(A) receptors using ultra fast perfusion techniques revealed a slower rate of the fast phase of desensitization compared with alpha1beta3gamma2S GABA(A) receptors. Additionally, current deactivation [a major determinant of inhibitory postsynaptic current (IPSC) duration] was faster in the mutated receptors. This raises the possibility of decreased GABAergic inhibition contributing to insomnia, as some members of the patient's family also suffer from insomnia. The mutation beta3(R192H) might, therefore, be linked to this condition. The intron/exon boundaries of the alpha1 subunit gene were also established and three additional variants were found in the alpha1 and beta3 genes.     

Functional anticodon architecture of human tRNALys3 includes disruption of intraloop hydrogen bonding by the naturally occurring amino acid modification, t6A

The structure of the human tRNA(Lys3) anticodon stem and loop domain (ASL(Lys3)) provides evidence of the physicochemical contributions of N6-threonylcarbamoyladenosine (t(6)A(37)) to tRNA(Lys3) functions. The t(6)A(37)-modified anticodon stem and loop domain of tRNA(Lys3)(UUU) (ASL(Lys3)(UUU)- t(6)A(37)) with a UUU anticodon is bound by the appropriately programmed ribosomes, but the unmodified ASL(Lys3)(UUU) is not [Yarian, C., Marszalek, M., Sochacka, E., Malkiewicz, A., Guenther, R., Miskiewicz, A., and Agris, P. F., Biochemistry 39, 13390-13395]. The structure, determined to an average rmsd of 1.57 +/- 0.33 A (relative to the mean structure) by NMR spectroscopy and restrained molecular dynamics, is the first reported of an RNA in which a naturally occurring hypermodified nucleoside was introduced by automated chemical synthesis. The ASL(Lys3)(UUU)-t(6)A(37) loop is significantly different than that of the unmodified ASL(Lys3)(UUU), although the five canonical base pairs of both ASL(Lys3)(UUU) stems are in the standard A-form of helical RNA. t(6)A(37), 3'-adjacent to the anticodon, adopts the form of a tricyclic nucleoside with an intraresidue H-bond and enhances base stacking on the 3'-side of the anticodon loop. Critically important to ribosome binding, incorporation of the modification negates formation of an intraloop U(33).A(37) base pair that is observed in the unmodified ASL(Lys3)(UUU). The anticodon wobble position U(34) nucleobase in ASL(Lys3)(UUU)-t(6)A(37) is significantly displaced from its position in the unmodified ASL and directed away from the codon-binding face of the loop resulting in only two anticodon bases for codon binding. This conformation is one explanation for ASL(Lys3)(UUU) tendency to prematurely terminate translation and -1 frame shift. At the pH 5.6 conditions of our structure determination, A(38) is protonated and positively charged in ASL(Lys3)(UUU)-t(6)A(37) and the unmodified ASL(Lys3)(UUU). The ionized carboxylic acid moiety of t(6)A(37) possibly neutralizes the positive charge of A(+)(38). The protonated A(+)(38) can base pair with C(32), but t(6)A(37) may weaken the interaction through steric interference. From these results, we conclude that ribosome binding cannot simply be an induced fit of the anticodon stem and loop, otherwise the unmodified ASL(Lys3)(UUU) would bind as well as ASL(Lys3)(UUU)-t(6)A(37). t(6)A(37) and other position 37 modifications produce the open, structured loop required for ribosomal binding.     

A frequent, naturally occurring mutation (P130T) of human hepatitis B virus core antigen is compensatory for immature secretion phenotype of another frequent variant (I97L)

A frequent mutation at codon 97 of human hepatitis B virus core antigen has been shown to cause an "immature secretion" phenotype, featuring nonselective and excessive secretions of virions containing immature viral genome. Our current study demonstrates that this abnormality can be efficiently offset by another frequent core mutation, P130T.     

Disulfide between Cys392 and Cys438 of human serum albumin is redox-active, which is responsible for the thioredoxin-supported lipid peroxidase activity

Human serum albumin (HSA) is an abundant protein found in blood plasma and extracellular fluids. Previously, we found that HSA has a distinct thioredoxin (Trx)-dependent lipid peroxidase activity in the presence of palmitoyl-CoA. In this paper, we identified the redox-active disulfide, which can be specifically reduced by Trx, responsible for the Trx-dependent lipid peroxidase activity. The IIB-III fragment of HSA (Pro299-Leu585) sustained the Trx-dependent lipid peroxidase activity. Chemical modification of the Trx-reduced IIB-III with a thiol-specific modification agent resulted in a complete loss of the peroxidase activity. The analysis of tryptic-peptides derived from the inactivated HSA and IIB-III revealed that Cys392 and Cys438, which exist as an intramolecular disulfide bond in HSA, were preferentially modified in both HSA and IIB-III. Taken together, these results suggested that HSA has a capability to reduce lipid hydroperoxide with the use of Trx as an in vivo electron donor, and that the redox-active disulfide between Cys392 and Cys438 acts as a primary site of the catalysis for the Trx-linked lipid peroxidase activity.     

Repulsive separation of the cytoplasmic ends of transmembrane helices 3 and 6 is linked to receptor activation in a novel thyrotropin receptor mutant (M626I)

Ligand-dependent activation of G protein-coupled receptors (GPCRs) involves repositioning of the juxtacytoplasmic ends of transmembrane helices TM3 and TM6. This concept, inferred from site-directed spin labeling studies, is supported by chemical cross-linking of the cytoplasmic ends of TM3 and TM6 blocking GPCR activation. Here we report a novel constitutive active mutation (M626I) in TM6 of the TSH receptor (TSHR), identified in affected members of a family with nonautoimmune hyperthyroidism. The specific constitutive activity of M626I, measured by its basal cAMP generation corrected for cell surface expression, was 13-fold higher than that of wild-type TSHR. Homology modeling of the TSHR serpentine domain based on the rhodopsin crystal structure suggests that M626 faces the side chain of I515 of TM3 near the membrane-cytoplasmic junction. Steric hindrance of the introduced isoleucine by I515 is consistent with the fact that shorter or more flexible side chains at position 626 did not increase constitutivity. Furthermore, a reciprocal mutation at position 515 (I515M), when introduced into the M626I background, acts as revertant mutation by allowing accommodation of the isoleucine sidechain at position 626 and fully restoring the constitutive activity to the level of wild-type TSHR. Thus, repulsive separation of the juxtacytoplasmic TM6 and TM3 in the M626I model conclusively demonstrates a direct link between the opening of this cytoplasmic face of the receptor structure and G protein coupling.     

Glu121-Lys319 salt bridge between catalytic and N-terminal domains is pivotal for the activity and stability of Escherichia coli aminopeptidase N

Escherichia coli aminopeptidase N (ePepN) belongs to the gluzincin family of M1 class metalloproteases that share a common primary structure with consensus zinc binding motif (HEXXH-(X18)-E) and an exopeptidase motif (GXMEN) in the active site. There is one amino acid, E121 in Domain I that blocks the extended active site grove of the thermolysin like catalytic domain (Domain II) limiting the substrate to S1 pocket. E121 forms a part of the S1 pocket, while making critical contact with the amino-terminus of the substrate. In addition, the carboxylate of E121 forms a salt bridge with K319 in Domain II. Both these residues are absolutely conserved in ePepN homologs. Analogous Glu-Asn pair in tricon interacting factor F3 (F3) and Gln-Asn pair in human leukotriene A(4) hydrolase (LTA(4) H) are also conserved in respective homologs. Mutation of either of these residues individually or together substantially reduced or entirely eliminated enzymatic activity. In addition, thermal denaturation studies suggest that the mutation at K319 destabilizes the protein as much as by 3.7 °C, while E121 mutants were insensitive. Crystal structure of E121Q mutant reveals that the enzyme is inactive due to the reduced S1 subsite volume. Together, data presented here suggests that ePepN, F3, and LTA(4) H homologs adopted a divergent evolution that includes E121-K319 or its analogous pairs, and these cannot be interchanged.     

T222P mutation of the insulin-like 3 hormone receptor LGR8 is associated with testicular maldescent and hinders receptor expression on the cell surface membrane

Insulin-like 3 (INSL3) hormone plays a crucial role in testicular descent during embryonic development. Genetic ablation of Insl3 or its G protein-coupled receptor (GPCR) Lgr8 causes cryptorchidism in mice. Previously, we identified a nonfunctional T222P mutation of LGR8 in several human patients with testicular maldescent. Using a large population of patients and healthy controls from Italy, we have demonstrated that T222P LGR8 mutation is present only in affected patients (19 T222P/+ of 598 vs. 0/450, P < 0.0001). We have also identified a novel allele of LGR8 (R223K) found in one patient with retractile testes. Both mutations are located in the leucine-rich repeats (LRRs) of GPCR ectodomain. The expression analysis of T222P mutant receptor transfected into 293T cells revealed that the mutation severely compromised GPCR cell membrane expression. The substitution of Thr(222) with the neutral Ser or Ala, or the R223K mutation, did not alter receptor cell membrane expression or ligand-induced cAMP increase. Additional mutations, affecting first leucine in a signature LxxLxLxxN/CxL stretch of LRR (L283F), or the amino acid residues, forming the disulfide bond or coordinating calcium ion in the LDLa module (C71Y and D70Y), also rendered proteins with reduced cell surface expression. The structural alterations of both LRRs and LDLa of the ligand-binding part of LGR8 cause the inability of receptor to express on the cell surface membrane and might be responsible for the abnormal testicular phenotype in patients.     

Marmoset red blood cell receptor for membrane-associated complement components is not related to human CR1: partial characterization of the C3-binding proteins responsible for the spontaneous rosette formation between marmoset red blood cells and human leukocytes

Cells from all the human B-lymphoblastoid cell lines tested and most human monocytes form rosettes with marmoset red blood cells (MaRBC). Because previous reports suggested the involvement of complement components in this phenomenon, the mechanism of rosette formation and the eventual similarities between the MaRBC receptor and the CR1 receptor present on human erythrocytes have been analyzed herein. The binding of MaRBC to human leukocytes strongly differs from the immune adherence phenomenon: rabbit anti-human CR1 did not react with MaRBC and the MaRBC receptor-binding activity is Ca2+-dependent. Rosette formation required intact energy metabolism and cytoskeleton integrity of leukocytes. Our attempts to purify the receptor from MaRBC membranes revealed the absence of CR1. Nevertheless, C3-binding proteins were isolated by selective desorption by Sepharose iC3 column chromatography. A three-band pattern was observed under reduced conditions with 74,000, 70,000, and 53,000 molecular weights. It was not possible to further separate these components. This protein complex inhibited the rosette phenomenon between MaRBC and both Raji and U-937 cells, exhibited a very poor cofactor activity, and had no decay-accelerating activity toward the classical C3 convertase. This material did not cross-react with antibodies directed to human proteins. These results showed that erythrocytes from new world monkeys do not express a receptor analogous to the human CR1, but expressed C3-binding protein with low cofactor activity that could recognize membrane-associated complement components.     

Chimeras of the rat and human FSH receptors (FSHRs) identify residues that permit or suppress transmembrane 6 mutation-induced constitutive activation of the FSHR via rearrangements of hydrophobic interactions between helices 6 and 7

Although a large number of naturally occurring activating mutations of the human LH receptor (hLHR) and human TSH receptor (hTSHR) have been identified, only one activating mutation of the human FSH receptor (hFSHR) has been found. Furthermore, mutations of several residues within the i3/transmembrane domain (TM) 6 region of the hFSHR that were done based upon known constitutively activating mutations of the human LHR were found to have no effect on hFSHR signaling. One of the hFSHR mutations examined in this context was the substitution of a highly conserved aspartate (D581) in TM6 with glycine. We show herein that although the basal activity of the rat FSHR (rFSHR) is similar to the hFSHR, mutation of the comparable residue (D580) in the rFSHR causes marked constitutive activation. Taking advantage of the high degree of amino acid identity between the rat and human FSHRs, we have used chimeras and point substitutions to determine the precise residues that suppress or permit constitutive activity by the D580/581G mutation. Thus, the simultaneous substitution of M576 in TM6 and H615 in TM7 of the hFSHR with the cognate rFSHR residues (threonine and tyrosine, respectively) now renders the hFSHR(D581G) mutant constitutively active. Conversely, the substitution of Y614 of the rFSHR with the cognate hFSHR residue (histidine) fully suppresses the constitutive activity of the rFSHR (D580G) mutant. Computer models of the human and rat FSHRs and mutants thereof were created based upon the crystal structure of rhodopsin. These models suggest that differences in hydrophobic interactions between TMs 6 and 7 of the rat and human FSHRs may account for the ability of TM6 of the rat, but not human, FSHR to adopt an active conformation as a result of the D580/581G mutation.     

The cysteine-rich domain of human T1R3 is necessary for the interaction between human T1R2-T1R3 sweet receptors and a sweet-tasting protein, thaumatin

Thaumatin is an intensely sweet-tasting protein perceived by humans but not rodents. Its threshold value of sweetness in humans is 50 nM, the lowest of any sweet-tasting protein. In the present study, the sites where sweet receptors interact with thaumatin were investigated using human embryonic kidney 293 (HEK293) cells expressing the sweet receptors T1R2–T1R3. Chimeric human– mouse sweet receptors were constructed and their responses to sweeteners were investigated. The human (h) T1R2– mouse (m) T1R3 combination responded to sucralose but not to thaumatin, clearly indicating that a T1R3 subunit from humans is necessary for the interaction with thaumatin. Furthermore, results obtained from using chimeric T1R3s showed that the cysteine-rich domain (CRD) of human T1R3 is important for the interaction with thaumatin. The CRD of T1R3 would be a prominent target for designing new sweeteners.