Robert Woodward

Investigation of the Role of Conserved Serine Residues in the Long Form of the Rat D2 Dopamine Receptor Using Site‐Directed Mutagenesis

Abstract: Three serine residues (Ser193, Ser194, Ser197) in the fifth transmembrane‐spanning region of the D2 dopamine receptor have been mutated separately to alanine and the effects of the mutations determined in ligand‐binding experiments with [3H]spiperone. For many antagonists the mutations had little effect, showing that the overall conformation of the mutant receptors was similar to that of the native, although there were effects on the binding of certain antagonists. The effect of the mutations on agonist binding to the free receptor (uncoupled from G proteins) was determined in the presence of GTP (100 µM). This showed that there was no single mode of binding of catecholamine agonists to the receptor and that all three serine residues can participate in the binding of some agonists, possibly through hydrogen bonds to the catechol hydroxyl groups. Coupling of the mutant receptors to G proteins was assessed from agonist‐binding curves in the absence of GTP, when higher and lower affinity agonist‐binding sites were seen. Receptor/G protein coupling was generally unaffected by the Ala193 and Ala194 mutations, but the Ala197 mutation eliminated receptor/G protein coupling for some agonists. These data show that the interactions of agonists with the free and coupled forms of the receptor are different.

Effect of Multiple Serine/Alanine Mutations in the Transmembrane Spanning Region V of the D2 Dopamine Receptor on Ligand Binding

Abstract: Three conserved serine residues (Ser193, Ser194, and Ser197) in transmembrane spanning region (TM) V of the D2 dopamine receptor have been mutated to alanine, individually and in combination, to explore their role in ligand binding and G protein coupling. The multiple Ser → Ala mutations had no effect on the binding of most antagonists tested, including [3H]spiperone, suggesting that the multiple mutations did not affect the overall conformation of the receptor protein. Double or triple mutants containing an Ala197 mutation showed a decrease in affinity for domperidone, whereas Ala193 mutants showed an increased affinity for a substituted benzamide, remoxipride. However, dopamine showed large decreases in affinity (>20‐fold) for each multiple mutant receptor containing the Ser193 Ala mutation, and the high‐affinity (coupled) state of the receptor (in the absence of GTP) could not be detected for any of the multiple mutants. A series of monohydroxylated phenylethylamines and aminotetralins was tested for their binding to the native and multiple mutant D2 dopamine receptors. The results obtained suggest that Ser193 interacts with the hydroxyl of S‐5‐hydroxy‐2‐dipropylaminotetralin (OH‐DPAT) and Ser197 with the hydroxyl of R‐5‐OH‐DPAT. We predict that Ser193 interacts with the hydroxyl of R‐7‐OH‐DPAT and the 3‐hydroxyl (m‐hydroxyl) of dopamine. Therefore, the conserved serine residues in TMV of the D2 dopamine receptor are involved in hydrogen bonding interactions with selected antagonists and most agonists tested and also enable agonists to stabilise receptor‐G protein coupling.

Molecular characterisation of a novel, repetitive protein of the paraflagellar rod in Trypanosoma brucei.

A partial cDNA clone, termed 5.20, was isolated from a lambda-gt11 phage expression library using a complex antiserum to the T. brucei cytoskeleton. Antisera against the fusion protein product of this 5.20 cDNA recognized a closely-spaced polypeptide doublet of high molecular weight (ca. 180-200 kDa) on immunoblots of T. brucei cytoskeletal preparations. Immunogold labelling suggested the 5.20 protein is intracellular and localized along the entire length of the paraflagellar rod. This pattern is similar to that generated with a monoclonal antibody, ROD1, which recognizes a high molecular weight protein doublet indistinguishable from that detected by 5.20-specific antisera. ROD1 recognizes mammalian spectrin, but the use of specific anti-spectrin antibodies for immunoblotting did not support ideas that 5.20 encodes spectrin or that spectrin can be specifically detected in T. brucei by such methods. Moreover, the sequence of the 5.20 cDNA insert bears little similarity, either in its nucleotide or predicted amino acid sequence to other known proteins and appears to be a unique cytoskeletal protein characterized especially by sequential amino acid sequence repetitiveness. The location of this novel protein suggests it may be responsible for providing either paraflagellar rod-membrane links or for organizing the more abundant paraflagellar rod structural proteins.

Structural Studies on D2 Dopamine Receptors: Mutation of a Histidine Residue Specifically Affects the Binding of a Subgroup of Substituted Benzamide Drugs

Abstract: A histidine residue (His394) that is likely to be located in the ligand‐binding region of the D2 dopamine receptor has been mutated to a leucine (Leu394), and the properties of the mutant receptor have been determined. For a range of antagonists the mutation has only a minor effect on the affinity of the receptor for the antagonist. The mutation does, however, elicit a structurally specific effect on the affinity with which certain members of the substituted benzamide class of antagonist bind to the receptor. Some of these drugs, e.g., sulpiride, sultopride, and tiapride, bind with reduced affinity to the mutated receptor, whereas others, e.g., clebopride and metoclopramide, bind with increased affinity. However, the Na+/H+ sensitivity of the binding of sulpiride to the receptor is not reduced by the mutation. These findings have been interpreted in terms of the productive or unfavourable interaction of the His394 residue with these compounds.

Immunological characterization of cytoskeletal proteins associated with the basal body, axoneme and flagellum attachment zone of Trypanosoma brucei

The monoclonal antibody BS7, raised to bovine sperm flagellum cytoskeletal antigens in a previous study, is here reported to detect flagellum-associated structures in Trypanosoma brucei and Crithidia fasciculata. Immunoblotting showed that BS7 cross-reacts with several cytoskeletal T. brucei proteins but phosphatase treatment did not diminish this complex immunoblot reactivity. To characterize further the cross-reactive proteins recognized in T. brucei-cytoskeletons by BS7 each was excised from preparative gels and used as an immunogen for antiserum production. Two proteins, with apparent sizes around 43 and 47 kDa, produced antisera shown to be monospecific by immunoblotting total T. brucei flagellum preparations. Each of these detected the basal body-associated immunofluorescence in T. brucei. Identification of the smaller, 43 kDa, component as a basal body-associated product was supported by the behaviour of a second monoclonal antibody, BBA4, which was also shown to detect the T. brucei basal body complex by immunofluorescence and immunoblots the 43 kDa polypeptide. These observations reveal new components of the trypanosome cytoskeleton. Also, they provide a further example of an immunological approach for identification of interesting, rare components of the T. brucei cytoskeleton starting from a complex mixture of proteins.

Isolation of cDNA clones encoding proteins of complex structures: analysis of the Trypanosoma brucei cytoskeleton

We have adapted a group of well-known procedures in order to devise a simple method that allows the isolation of specific cDNAs encoding proteins located in different regions of the Trypanosoma brucei cytoskeleton. cDNA clones were isolated by screening a lambda gt11 expression library with a polyspecific, polyclonal antiserum against a complex immunogen, in this case the complete cytoskeleton. The fusion proteins produced by the clones were then used as an affinity immunoadsorbant to select monospecific polyclonals. The monospecific antisera isolated were used as probes to identify and localize different cytoskeleton proteins by Western blotting and immunofluorescence. This method proved particularly useful for the molecular identification of minor components in a complex structure. It should prove applicable to the molecular analysis of other organelles or protein complexes.