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PubMed1990

Production of interspecies chimeric calves by aggregation of Bos indicus and Bos taurus demi-embryos.

Two experiments were conducted to produce interspecific chimeric calves by aggregation of B. indicus and B. taurus demi-embryos. In the first experiment, morulae, compacted morulae, and early blastocysts were collected nonsurgically from Brahman (B. indicus) and Friesian (B. taurus) donors. Embryos were bisected and one demi-embryo from each species was placed in a single zone pellucida. In the second experiment, Brahman (B. indicus) and Hereford-Shorthorn (HS) (B. taurus) demi-embryos were aggregated. The resulting 'chimeric embryos' were transferred nonsurgically to synchronous recipients immediately following microsurgery. Of 112 recipients of 112 chimeric embryos, 29 (26%) were pregnant at 60 days. Of these, 24 (83%) produced full-term calves and 5 (17%) aborted between 2 and 5 months' gestation. From the 24 full-term pregnancies, two sets of twins and 22 singleton calves were born. Of the 22 singletons, 15 were chimeric including six bull calves (one Brahman-Friesian and five Brahman-HS) that were overt chimeras. All the overt chimeras resulted from aggregation of halves of early morulae (precompaction).

Animals↗
PubMed1984

Chimeric influenza virus hemagglutinin containing either the NH2 terminus or the COOH terminus of G protein of vesicular stomatitis virus is defective in transport to the cell surface.

Chimeric cDNA clones of influenza virus hemagglutinin (HA) were constructed in which the DNA encoding either the NH2 terminus or the COOH terminus of HA was replaced with that of a vesicular stomatitis virus G protein. The chimeric cDNAs (GHA or HAG) were expressed in CV1 cells using the simian virus 40 late replacement promoter. Both chimeric proteins are synthesized, glycosylated, and transported to the rough endoplasmic reticulum. These results show that the NH2-terminal sequences of vesicular stomatitis virus G protein can provide a signal function for translocation and the COOH-terminal sequences can provide the anchor function for the influenza virus HA, when substituted for similar sequences. However, the chimeric glycoproteins were not transported to the Golgi complex or the plasma membrane. The implication of these results in translocation, sorting, and transport processes is discussed.

Animals↗
PubMed1987

Secretion of a chimeric T-cell receptor-immunoglobulin protein.

To produce sufficient quantities of soluble T-cell receptor protein for detailed biochemical and biophysical analyses we have explored the use of immunoglobulin--T-cell receptor gene fusions. In this report we describe a chimeric gene construct containing a T-cell receptor alpha-chain variable (V) domain and the constant (C) region coding sequences of an immunoglobulin gamma 2a molecule. Cells transfected with the chimeric gene synthesize a stable protein product that expresses immunoglobulin and T-cell receptor antigenic determinants as well as protein A binding sites. We show that the determinant recognized by the anticlonotypic antibody A2B4.2 resides on the V alpha domain of the T-cell receptor. The chimeric protein associates with a normal lambda light chain to form an apparently normal tetrameric (H2L2, where H = heavy and L = light) immunoglobulin molecule that is secreted. Also of potential significance is the fact that a T-cell receptor V beta gene in the same construct is neither assembled nor secreted with the lambda light chain, and when expressed with a C kappa region it does not assemble with the chimeric V alpha C gamma 2a protein mentioned above. This indicates that not all T-cell receptor V regions are similar enough to immunoglobulin V regions for them to be completely interchangeable.

Amino Acid Sequence↗
PubMed1989

Mouse/human chimeric monoclonal antibody in man: kinetics and immune response.

A mouse/human chimeric monoclonal antibody (mAb) composed of the variable regions of murine 17-1A mAb and the constant regions of human IgG-1K immunoglobulin was administered to 10 patients with metastatic colon cancer. Four patients received single infusion of 10 mg (n = 2) or 40 mg (n = 2). Six patients received three infusion of 10 mg (n = 3) or 40 mg (n = 3) at 2-week intervals. The pharmacokinetics were similar at both dose levels and at the second and third infusions. The plasma disappearance curves were best fit by a two-compartment model having a mean alpha T1/2 of 17.5 hr (range 13-23 hr) and a mean beta T1/2 of 100.5 hr (range 65-139 hr). One patient who received three 40-mg doses of chimeric IgG-1K 17-1A mAb (day 0, 14, and 28) was the only patient to exhibit a detectable but modest antibody reactivity to chimera on days 63 and 84. The antibody reactivity was inhibited by murine 17-1A mAb, indicating that the antibody response was directed to the murine variable region of the chimera. The patients had no toxic or allergic reactions. This chimeric form of 17-1A mAb has an approximate 6-fold longer circulation time and appears to be substantially less immunogenic than its murine counterpart. These characteristics may provide an advantage in the clinical application of such chimeric molecules in therapeutic trials in humans.

Adenocarcinoma↗
PubMed1989

Expression of immunoglobulin-T-cell receptor chimeric molecules as functional receptors with antibody-type specificity.

To design and direct at will the specificity of T cells in a non-major histocompatibility complex (MHC)-restricted manner, we have generated and expressed chimeric T-cell receptor (TcR) genes composed of the TcR constant (C) domains fused to the antibody's variable (V) domains. Genomic expression vectors have been constructed containing the rearranged gene segments coding for the V region domains of the heavy (VH) and light (VL) chains of an anti-2,4,6-trinitrophenyl (TNP) antibody (SP6) spliced to either one of the C-region gene segments of the alpha or beta TcR chains. Following transfection into a cytotoxic T-cell hybridoma, expression of a functional TcR was detected. The chimeric TcR exhibited the idiotope of the Sp6 anti-TNP antibody and endowed the T cells with a non-MHC-restricted response to the hapten TNP. The transfectants specifically killed and produced interleukin 2 in response to TNP-bearing target cells across strain and species barriers. Moreover, such transfectants responded to immobilized TNP-protein conjugates, bypassing the need for cellular processing and presentation. In the particular system employed, both the TNP-binding site and the Sp6 idiotope reside almost exclusively in the VH chain region. Hence, introduction into T cells of TcR genes containing only the VHSp6 fused to either the C alpha or C beta was sufficient for the expression of a functional surface receptor. Apparently, the VHC alpha or VHC beta chimeric chains can pair with the endogenous beta or alpha chains of the recipient T cell to form a functional alpha beta heterodimeric receptor. Thus, this chimeric receptor provides the T cell with an antibody-like specificity and is able to effectively transmit the signal for T-cell activation and execution of its effector function.

Animals↗
PubMed1992

Construction and characterization of chimeric tick-borne encephalitis/dengue type 4 viruses.

Dengue type 4 virus (DEN4) cDNA was used as a vector to express genes of the distantly related tick-borne encephalitis virus (TBEV). Full-length chimeric TBEV/DEN4 cDNAs were constructed by substituting TBEV genes coding for proteins such as capsid (C); pre-membrane, which is the precursor of membrane (M); envelope (E); or nonstructural protein NS1 for the corresponding DEN4 sequences. RNA transcripts prepared from cDNAs were used to transfect permissive simian cells. Two viable chimeric viruses that contained TBEV CME or ME genes were recovered. Compared with DEN4, chimeric TBE(ME)/DEN4 virus [designated vTBE(ME)/DEN4] produced larger plaques and grew to higher titer in simian cells. In contrast, vTBE(ME)/DEN4 produced smaller plaques on mosquito cells and grew to lower titer than DEN4. Analysis of viral RNA and proteins produced in vTBE(ME)/DEN4- and DEN4-infected mosquito or simian cells revealed that the chimera was restricted in its ability to enter and replicate in mosquito cells. In contrast, vTBE(ME)/DEN4 entered simian cells efficiently and its RNA was replicated more rapidly in these cells than was parental DEN4 RNA. Following intracerebral inoculation, vTBE(ME)/DEN4 caused fatal encephalitis in both suckling and adult mice, while nearly all mice inoculated by the same route with DEN4 did not develop disease. Unlike wild-type TBEV, vTBE(ME)/DEN4 did not cause encephalitis when adult mice were inoculated by a peripheral route. Adult mice previously inoculated with the chimera by a peripheral route were completely resistant to subsequent intraperitoneal challenge with 10(3) times the median lethal dose of TBEV, whereas mice previously inoculated with DEN4 were not protected. These findings indicate that (i) the TBEV M and E genes of the chimeric virus are major protective antigens and induce resistance to lethal TBEV challenge and (ii) other regions of the TBEV genome are essential for the ability of this virus to spread from a peripheral site to the brain. Success in constructing a viable TBEV/DEN4 chimera that retains the protective antigens of TBEV but lacks its peripheral invasiveness provides a strategy for the development of live attenuated TBEV vaccines.

Amino Acid Sequence↗
PubMed1993

Specific activation and targeting of cytotoxic lymphocytes through chimeric single chains consisting of antibody-binding domains and the gamma or zeta subunits of the immunoglobulin and T-cell receptors.

The generation of tumor-specific lymphocytes and their use in adoptive immunotherapy is limited to a few malignancies because most spontaneous tumors are very weak or not at all immunogenic. On the other hand, many anti-tumor antibodies have been described which bind tumor-associated antigens shared among tumors of the same histology. Combining the variable regions (Fv) of an antibody with the constant regions of the T-cell receptor (TCR) chains results in chimeric genes endowing T lymphocytes with antibody-type specificity, potentially allowing cellular adoptive immunotherapy against types of tumors not previously possible. To generalize and extend this approach to additional lymphocyte-activating molecules, we designed and constructed chimeric genes composed of a single-chain Fv domain (scFv) of an antibody linked with gamma or zeta chains, the common signal-transducing subunits of the immunoglobulin receptor and the TCR. Such chimeric genes containing the Fv region of an anti-trinitophenyl antibody could be expressed as functional surface receptors in a cytolytic T-cell hybridoma. They triggered interleukin 2 secretion upon encountering antigen and mediated non-major-histocompatibility-complex-restricted hapten-specific target cell lysis. Such chimeric receptors can be exploited to provide T cells and other effector lymphocytes, such as natural killer cells, with antibody-type recognition directly coupled to cellular activation.

Animals↗
PubMed1993

High-frequency plastid transformation in tobacco by selection for a chimeric aadA gene.

We report here a 100-fold increased frequency of plastid transformation in tobacco by selection for a chimeric aadA gene encoding aminoglycoside 3"-adenylyltransferase, as compared with that obtained with mutant 16S rRNA genes. Expression of aadA confers resistance to spectinomycin and streptomycin. In transforming plasmid pZS197, a chimeric aadA is cloned between rbcL and open reading frame ORF512 plastid gene sequences. Selection was for spectinomycin resistance after biolistic delivery of pZS197 DNA into leaf cells. DNA gel-blot analysis confirmed incorporation of the chimeric aadA gene into the plastid genome by two homologous recombination events via the flanking plastid gene sequences. The chimeric gene became homoplasmic in the recipient cells and is uniformly transmitted to the maternal seed progeny. The ability to transform routinely plastids of land plants opens the way to manipulate the process of photosynthesis and to incorporate novel genes into the plastid genome of crops.

Base Sequence↗
PubMed1993

Dexamethasone negatively regulates the activity of a chimeric dihydrofolate reductase/glucocorticoid receptor protein.

A chimeric gene was constructed encoding the entire murine dihydrofolate reductase (DHFR) protein with a carboxyl-terminal extension encompassing amino acids 494-795 of the rat glucocorticoid receptor (GR). The chimeric DHFR/GR gene encoded a functional DHFR protein, as measured by the ability to transform DHFR-deficient Chinese hamster ovary (CHO) cells to a DHFR-positive phenotype. The DHFR/GR protein bound [3H]dexamethasone with a similar affinity as wild-type GR. Selection of stable CHO transformants in increasing concentrations of methotrexate resulted in increased expression of DHFR/GR. Addition of dexamethasone, a synthetic glucocorticoid agonist, decreased the activity of the chimeric protein, as measured by colony formation in selective medium, binding of fluoresceinated methotrexate, and direct enzymatic assay for DHFR. Addition of RU486, a glucocorticoid antagonist, antagonized the effect of dexamethasone. In the absence of dexamethasone, the chimeric protein was primarily localized to the cytoplasm. In the presence of dexamethasone or RU486, DHFR/GR translocated into the nucleus. However, RU486 did not decrease DHFR activity, distinguishing subcellular location from functional activity. These results demonstrate that glucocorticoids negatively affect the function of DHFR/GR.

Animals↗
PubMed1997

Development of a novel anti-HIV-1 agent from within: effect of chimeric Vpr-containing protease cleavage site residues on virus replication.

Effective antiviral agents will be of great value in controlling virus replication and delaying the onset of HIV-1-related disease symptoms. Current therapy involves the use of antiviral agents that target the enzymatic functions of the virus, resulting in the emergence of resistant viruses to these agents, thus lowering their effectiveness. To overcome this problem, we have considered the idea of developing novel agents from within HIV-1 as inhibitors of virus replication. The specificity of the Vpr protein for the HIV-1 virus particle makes it an attractive molecule for the development of antiviral agents targeting the events associated with virus maturation. We have generated chimeric Vpr proteins containing HIV-1-specific sequences added to the C terminus of Vpr. These sequences correspond to nine cleavage sites of the Gag and Gag-Pol precursors of HIV-1. The chimeric Vpr constructs were introduced into HIV-1 proviral DNA to assess their effect on virus infectivity using single- and multiple-round replication assays. The virus particles generated exhibited a variable replication pattern depending on the protease cleavage site used as a fusion partner. Interestingly, the chimeric Vpr containing the cleavage sequences from the junction of p24 and p2, 24/2, completely abolished virus infectivity. These results show that chimeric proteins generated from within HIV-1 have the ability to suppress HIV-1 replication and make ideal agents for gene therapy or intracellular immunization to treat HIV-1 infection.

Anti-HIV Agents↗
PubMed1995

Structure and function of several anti-dansyl chimeric antibodies formed by domain interchanges between human IgM and mouse IgG2b.

Two pairs of chimeric, domain-switched immunoglobulins with identical murine, anti-dansyl (5-dimethylaminonaphthalene-1-sulfonyl) variable domains have been generated, employing as parent antibodies a human IgM and a mouse IgG2b. The first pair of chimeric antibodies mu mu gamma mu and gamma gamma mu gamma was generated by switching the C mu 3 and C gamma 2 domains between IgM and IgG2b. The second pair of chimeras mu mu gamma gamma and gamma gamma mu mu were formed by switching both C mu 3 and C mu 4 with C gamma 2 and C gamma 3. SDS-polyacrylamide gel electrophoresis and analytical ultracentrifugation showed that over half (57 and 71%) of the two chimeric antibodies possessing the C mu 4 domain and tail piece formed disulfide-linked IgM-like polymers. In contrast, the two chimeric antibodies lacking the C mu 4 domain were almost entirely monomeric. Both monomeric chimeras had reduced ability to activate complement. The chimera gamma gamma mu gamma had no activity under any of the assay conditions, whereas mu mu gamma gamma caused only a small amount of cell lysis but was fully active in consuming complement at 4 degrees C. The polymeric chimera gamma gamma mu mu was much less active than IgM, bound C1 weakly and caused some cell lysis but consumed little complement with soluble antigen. The polymeric chimera mu mu gamma mu bound C1 strongly and was the most active antibody in all assays, even more active than the parental IgG2b and IgM antibodies; it was the only antibody that exhibited antigen-independent activity. The results suggest that C mu 3 alone does not constitute the complement binding site in IgM but requires both C mu 1-2 and C mu 4 for full activity.

Animals↗
PubMed1997

Identification of domains conferring ligand binding specificity to the prostanoid receptor. Studies on chimeric prostacyclin/prostaglandin D receptors.

To identify domains conferring ligand binding specificity to prostanoid receptors, we constructed a series of chimeric receptors by successively replacing the regions from the carboxyl-terminal tail of mouse prostacyclin (prostaglandin I (PGI)) receptor (mIP) with the corresponding regions of the mouse PGD receptor (mDP). The mIP receptor expressed in COS 7 cells bound [3H]iloprost, a PGI2 analog, and [3H]PGE1 with Kd values of 13 and 27 nM, respectively. This receptor did not bind [3H]PGD2, [3H]PGE2, and [3H]PGF2alpha. The mDP receptor bound only [3H]PGD2 with a Kd value of 43 nM. The chimeric IPN-VII/DPC receptor with replacement of the carboxyl tail of the mIP receptor with that of the mDP receptor showed 12-16-fold higher affinities for [3H]iloprost and [3H]PGE1 than the mIP receptor. The region extending from the sixth transmembrane domain to the carboxyl terminus of the mIP receptor was next replaced with the corresponding region of the mDP receptor. This chimeric IPN-V/DPVI-C receptor acquired the ability to bind [3H]PGD2 and [3H]PGE2 without decreasing the affinities of the mIP receptor to [3H]iloprost and [3H]PGE1. These binding characteristics did not change when the fourth and fifth transmembrane domains of the mIP receptor were further replaced with the corresponding regions of the mDP receptor. However, when the first extracellular to second intracellular loop of the mIP receptor containing the third transmembrane domain was further replaced with those of the mDP receptor, the affinities for [3H]PGE1, [3H]PGE2, and [3H]iloprost were markedly decreased, whereas that for [3H]PGD2 was increased by about 2-fold. [3H]PGF2alpha showed no affinity for the mIP, mDP, and all the chimeric receptors. These results suggest that the sixth to seventh transmembrane domain of the mIP receptor confers the specificity of this receptor to bind selectively to PGE1 and not to PGE2 and that the third transmembrane domain of the mDP receptor confers the selective binding of PGD2 to this receptor.

Animals↗
PubMed1997

Construction of chimeric enzymes out of maize endosperm branching enzymes I and II: activity and properties.

Branching enzyme I and II isoforms from maize endosperm (mBE I and mBE II, respectively) have quite different properties, and to elucidate the domain(s) that determines the differences, chimeric genes consisting of part mBE I and part mBE II were constructed. When expressed under the control of the T7 promoter in Escherichia coli, several of the chimeric enzymes were inactive. The only fully active chimeric enzyme was mBE II-I BspHI, in which the carboxyl-terminal part of mBE II was exchanged for that of mBE I at a BspHI restriction site and was purified to homogeneity and characterized. Another chimeric enzyme, mBE I-II HindIII, in which the amino-terminal end of mBE II was replaced with that of mBE I, had very little activity and was only partially characterized. The purified mBE II-I BspHI exhibited higher activity than wild-type mBE I and mBE II when assayed by the phosphorylase a stimulation assay. mBE II-I BspHI had substrate specificity (preference for amylose rather than amylopectin) and catalytic capacity similar to mBE I, despite the fact that only the carboxyl terminus was from mBE I, suggesting that the carboxyl terminus may be involved in determining substrate specificity and catalytic capacity. In chain transfer experiments, mBE II-I BspHI transferred more short chains (with a degree of polymerization of around 6) in a fashion similar to mBE II. In contrast, mBE I-II HindIII transferred more long chains (with a degree of polymerization of around 11-12), similar to mBE I, suggesting that the amino terminus of mBEs may play a role in the size of oligosaccharide chain transferred. This study challenges the notion that the catalytic centers for branching enzymes are exclusively located in the central portion of the enzyme; it suggests instead that the amino and carboxyl termini may also be involved in determining substrate preference, catalytic capacity, and chain length transfer.

1,4-alpha-Glucan Branching Enzyme↗
PubMed1998

Probing domain functions of chimeric PDE6alpha'/PDE5 cGMP-phosphodiesterase.

Chimeric cGMP phosphodiesterases (PDEs) have been constructed using components of the cGMP-binding PDE (PDE5) and cone photoreceptor phosphodiesterase (PDE6alpha') in order to study structure and function of the photoreceptor enzyme. A fully functional chimeric PDE6alpha'/PDE5 enzyme containing the PDE6alpha' noncatalytic cGMP-binding sites, and the PDE5 catalytic domain has been efficiently expressed in the baculovirus/High Five cell system. The catalytic properties of this chimera were practically indistinguishable from those of PDE5, whereas the noncatalytic cGMP binding was similar to that of native purified PDE6alpha'. The inhibitory gamma subunit of PDE6 (Pgamma) enhanced the affinity of cGMP binding at noncatalytic sites of native PDE6alpha' by approximately 6-fold. The polycationic region of Pgamma, Pgamma-24-45, was mainly responsible for this effect, while the inhibitory domain of Pgamma, Pgamma-63-87, was ineffective. On the contrary, Pgamma failed to inhibit catalytic activity of the chimeric PDE6alpha'/PDE5 or to modulate its noncatalytic cGMP binding. Substitutions of Ala residues for the conserved Asn, Asn193 or Asn402, in the two N(K/R)XD-like motifs of the chimeric PDE noncatalytic cGMP-binding sites, each led to a loss of the noncatalytic cGMP binding. Our data suggest that both putative noncatalytic sites of PDE6alpha' are important for binding of cGMP, and that the two binding sites are coupled. Furthermore, mutation Asn402 --> Ala resulted in an approximately 10-fold increase of the Km value for cGMP, indicating that occupation of the noncatalytic cGMP- binding sites of PDE6alpha' may regulate catalytic properties of the enzyme.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1999

Chimeric analysis of a neuronal nicotinic acetylcholine receptor reveals amino acids conferring sensitivity to alpha-bungarotoxin.

We have investigated the molecular determinants responsible for alpha-bungarotoxin (alphaBgtx) binding to nicotinic acetylcholine receptors through chimeric analysis of two homologous alpha subunits, one highly sensitive to alphaBgtx block (alpha1) and the other, alphaBgtx-insensitive (alpha3). By replacing rat alpha3 residues 184-191 with the corresponding region from the Torpedo alpha1 subunit, we introduced a cluster of five alpha1 residues (Trp-184, Trp-187, Val-188, Tyr-189, and Thr-191) into the alpha3 subunit. Functional activity and alphaBgtx sensitivity were assessed following co-expression in Xenopus oocytes of the chimeric alpha3 subunit (alpha3/alpha1[5]) with either rat beta2 or beta4 subunits. Agonist-evoked responses of alpha3/alpha1[5]-containing receptors were blocked by alphaBgtx with nanomolar affinity (IC(50) values: 41 nM for alpha3/alpha1[5]beta2 and 19 nM for alpha3/alpha1[5]beta4). Furthermore, receptors containing the single point mutation alpha3K189Y acquire significant sensitivity to alphaBgtx block (IC(50) values: 186 nM for alpha3K189Ybeta2 and 179 nM for alpha3K189Ybeta4). Another alpha3 chimeric subunit, alpha3/alpha7[6], similar to alpha3/alpha1[5] but incorporating the corresponding residues from the alphaBgtx-sensitive alpha7 subunit, also conferred potent alphaBgtx sensitivity to chimeric receptors when co-expressed with the beta4 subunit (IC(50) value = 31 nM). Our findings demonstrate that the residues between positions 184 and 191 of the alphaBgtx-sensitive subunits alpha1 and alpha7 play a critical functional role in the interaction of alphaBgtx with nicotinic acetylcholine receptors sensitive to this toxin.

Amino Acid Sequence↗
PubMed1999

Catabolism of asialo-GM2 in man and mouse. Specificity of human/mouse chimeric GM2 activator proteins.

Tay-Sachs disease is an inborn lysosomal disease characterized by excessive cerebral accumulation of GM2. The catabolism of GM2 to GM3 in man requires beta-hexosaminidase A (HexA) and a protein cofactor, the GM2 activator. Thus, Tay-Sachs disease can be caused by the deficiency of either HexA or the GM2 activator. The same cofactor found in mouse shares 74.1% amino acid identity (67% nucleotide identity) with the human counterpart. Between the two activators, the mouse GM2 activator can effectively stimulate the hydrolysis of both GM2 and asialo-GM2 (GA2) by HexA and, to a lesser extent, also stimulate HexB to hydrolyze GA2, whereas the human activator is ineffective in stimulating the hydrolysis of GA2 (Yuziuk, J. A., Bertoni, C., Beccari, T., Orlacchio, A., Wu, Y.-Y., Li, S.-C., and Li, Y.-T. (1998) J. Biol. Chem. 273, 66-72). To understand the role of these two activators in stimulating the hydrolyses of GM2 and GA2, we have constructed human/mouse chimeric GM2 activators and studied their specificities. We have identified a narrow region (Asn(106)-Tyr(114)) in the mouse cDNA sequence that might be responsible for stimulating the hydrolysis of GA2. Replacement of the corresponding site in the human sequence with the specific mouse sequence converted the ineffective human activator into an effective chimeric protein for stimulating the hydrolysis of GA2. This chimeric activator protein, like the mouse protein, is also able to stimulate the hydrolysis of GA2 by HexB. The mouse model of human type B Tay-Sachs disease recently engineered by the targeted disruption of the Hexa gene showed less severe clinical manifestation than found in human patients. This has been considered to be the result of the catabolism of GM2 via converting it to GA2 and further hydrolysis of GA2 to lactosylceramide by HexB with the assistance of mouse GM2 activator protein. The chimeric activator protein that bears the characteristics of the mouse GM2 activator may therefore be able to induce an alternative catabolic pathway for GM2 in human type B Tay-Sachs patients.

Amino Acid Sequence↗
PubMed2000

Chimeric melatonin mt1 and melatonin-related receptors. Identification of domains and residues participating in ligand binding and receptor activation of the melatonin mt1 receptor.

Melatonin receptors bind and become activated by melatonin. The melatonin-related receptor, despite sharing considerable amino acid sequence identity with melatonin receptors, does not bind melatonin and is currently an orphan G protein-coupled receptor. To investigate the structure and function of both receptors, we engineered a series of 14 chimeric receptor constructs, allowing us to determine the relative contribution of each transmembrane domain to ligand binding and receptor function. Results identified that when sequences encoding transmembrane domains 1, 2, 3, 5, or 7 of the melatonin mt(1) receptor were replaced by the corresponding domains of the melatonin-related receptor, the resultant chimeric receptors all displayed specific 2-[(125)I]iodomelatonin binding. Replacement of sequences incorporating transmembrane domains 4 or 6, however, resulted in chimeric receptors that displayed no detectable 2-[(125)I]iodomelatonin binding. The subsequent testing of a "reverse" chimeric receptor in which sequences encoding transmembrane domains 4 and 6 of the melatonin-related receptor were replaced by the corresponding melatonin mt(1) receptor sequences identified specific 2-[(125)I]iodomelatonin binding and melatonin-mediated modulation of cyclic AMP levels. To further investigate these findings, site-directed mutagenesis was performed on residues within transmembrane domain 6 of the melatonin mt(1) receptor. This identified Gly(258) (Gly(6.55)) as a critical residue required for high affinity ligand binding and receptor function.

Animals↗
PubMed2001

Casein kinase II sites in the intracellular C-terminal domain of the thyrotropin-releasing hormone receptor and chimeric gonadotropin-releasing hormone receptors contribute to beta-arrestin-dependent internalization.

We have previously shown that the mammalian gonadotropin-releasing hormone receptor (GnRHR), a unique G-protein-coupled receptor (GPCR) lacking an intracellular carboxyl tail (C-tail), does not follow a beta-arrestin-dependent internalization pathway. However, internalization of a chimeric GnRHR with the thyrotropin-releasing hormone receptor (TRHR) C-tail does utilize beta-arrestin. Here, we have investigated the sites within the intracellular C-tail domain that are important for conferring beta-arrestin-dependent internalization. In contrast to the chimeric GnRHR with a TRHR C-tail, a chimeric GnRHR with the catfish GnRHR C-tail is not beta-arrestin-dependent. Sequence comparisons between these chimeric receptors show three consensus phosphorylation sites for casein kinase II (CKII) in the TRHR C-tail but none in the catfish GnRHR C-tail. We thus investigated a role for CKII sites in determining GPCR internalization via beta-arrestin. Sequential introduction of three CKII sites into the chimera with the catfish C-tail (H354D,A366E,G371D) resulted in a change in the pattern of receptor phosphorylation and beta-arrestin-dependence, which only occurred when all three sites were introduced. Conversely, mutation of the putative CKII sites (T365A,T371A,S383A) in the C-tail of a beta-arrestin-sensitive GPCR, the TRHR, resulted in decreased receptor phosphorylation and a loss of beta-arrestin-dependence. Mutation of all three CKII sites was necessary before a loss of beta-arrestin-dependence was observed. Visualization of beta-arrestin/GFP redistribution confirmed a loss or gain of beta-arrestin sensitivity for receptor mutants. Internalization of receptors without C-tail CKII sites was promoted by a phosphorylation-independent beta-arrestin mutant (R169E), suggesting that these receptors do not contain the necessary phosphorylation sites required for beta-arrestin-dependent internalization. Apigenin, a specific CKII inhibitor, blocked the increase in receptor internalization by beta-arrestin, thus providing further support for the involvement of CKII. This study presents evidence of a novel role for C-tail CKII consensus sites in targeting these GPCRs to the beta-arrestin-dependent pathway.

Amino Acid Sequence↗