Papers by Colleen Flanagan
Journal of Biological Chemistry, 2001
Mammalian receptors for gonadotropin-releasing hormone (GnRH) have over 85% sequence homology and... more Mammalian receptors for gonadotropin-releasing hormone (GnRH) have over 85% sequence homology and similar ligand selectivity. Biological studies indicated that the chicken GnRH receptor has a distinct pharmacology, and certain antagonists of mammalian GnRH receptors function as agonists. To explore the structural determinants of this, we have cloned a chicken pituitary GnRH receptor and demonstrated that it has marked differences in primary amino acid sequence (59% homology) and in its interactions with GnRH analogs. The chicken GnRH receptor had high affinity for mammalian GnRH (K(i) 4.1 +/- 1.2 nM), similar to the human receptor (K(i) 4.8 +/- 1.2 nM). But, in contrast to the human receptor, it also had high affinity for chicken GnRH ([Gln(8)]GnRH) and GnRH II ([His(5),Trp(7),Tyr(8)]GnRH) (K(i) 5.3 +/- 0.5 and 0.6 +/- 0.01 nM). Three mammalian receptor antagonists were also pure antagonists in the chicken GnRH receptor. Another three, characterized by D-Lys(6) or D-isopropyl-Lys(6) moieties, functioned as pure antagonists in the human receptor but were full or partial agonists in the chicken receptor. This suggests that the Lys side chain interacts with functional groups of the chicken GnRH receptor to stabilize it in the active conformation and that these groups are not available in the activated human GnRH receptor. Substitution of the human receptor extracellular loop two with the chicken extracellular loop two identified this domain as capable of conferring agonist activity to mammalian antagonists. Although functioning of antagonists as agonists has been shown to be species-dependent for several GPCRs, the dependence of this on an extracellular domain has not been described.
AIDS Research and Human Retroviruses, 2008
HIV-1 subtype C is the fastest spreading subtype worldwide and predominantly uses the CCR5 corece... more HIV-1 subtype C is the fastest spreading subtype worldwide and predominantly uses the CCR5 coreceptor, showing minimal transition to the X4 phenotype. This raises the possibility that envelope proteins of HIV-1 subtype C have structural features that favor interaction with CCR5. Preference for CCR5 could arise from enhanced affinity of HIV-1 subtype C for CCR5. To test this, we have characterized the interaction of gp120 envelope proteins from HIV-1 subtype C clones with CD4 and CCR5. Recombinant gp120 proteins from isolates of HIV-1 subtypes B and C were expressed, purified, and assessed in a CD4 binding assay and a CCR5 chemokine competition binding assay. All gp120 proteins bound to CD4-expressing cells, except one, 97ZA347ts, which had Arg substituted for the Cys239 in the conserved C2 loop. Reconstitution of Cys239, using site-directed mutagenesis, restored CD4 binding, while introducing Arg or Ser into position 239 of the functional Du151 gp120 protein abrogated CD4 binding. This shows that the Cys228-Cys239 disulfide bond of gp120 is required for high-affinity binding to CD4. Recombinant gp120 proteins from two HIV-1 subtype B clones bound CCR5 in the presence of CD4, while gp120 from the X4-tropic, HxB2, clone did not bind CCR5. gp120 from two functional HIV-1 subtype C clones, Du151 and MOLE1, bound CCR5 with high affinity in the presence of CD4 and Du151 showed significant CCR5 binding in the absence of CD4. A gp120 from a nonfunctional subtype C clone had lower affinity for CCR5. These results indicate that HIV-1 subtype C proteins have high affinity for CCR5 with variable dependence on CD4.
Molecular and Cellular Endocrinology, 2004
Mammalian gonadotropin releasing hormone (GnRH) receptors have a conserved acidic residue (Glu7.3... more Mammalian gonadotropin releasing hormone (GnRH) receptors have a conserved acidic residue (Glu7.32(301) or Asp7.32(302)) in extracellular loop (ECL) three that confers selectivity for mammalian GnRH, which has Arg8. Comparison of mammalian and non-mammalian GnRH receptors suggested that the acidic residue is not the only determinant of ligand selectivity in mammalian receptors. The acidic residue is followed by a conserved Pro7.33 in mammalian GnRH receptors, but not non-mammalian receptors. Unique structural constraints imposed by Pro residues suggested that Pro7.33 determines selective binding of Arg8-containing GnRH, by stabilising the conformation of the third extracellular loop of the receptor. Substituting Pro7.33(303) or introducing Pro to position 7.31 decreased affinity for GnRH, but not analogs lacking Arg8. Substituting Pro7.33(303) changed the predicted alpha-helix content of the loop-helix interface. These results show that Pro7.33(303) of the human GnRH receptor is required for selective high affinity binding of mammalian GnRH and supports the hypothesis that Pro7.33(303) stabilises a loop conformation that is necessary for selective ligand binding.
Biochemical and Biophysical Research Communications, 1993
The cloned sheep gonadotropin-releasing hormone (GnRH) receptor was analysed for sequence homolog... more The cloned sheep gonadotropin-releasing hormone (GnRH) receptor was analysed for sequence homology/differences among mammalian receptors and its pharmacology characterized in COS-1 cells. Transmembrane domains TM2, TM3, TM5, TM6 and TM7, and extracellular loop 1 are most highly conserved (> 90%) in this G-protein coupled receptor. The Kd of the sheep receptor in binding assays (4.9 nM) was similar to the human and rat receptors, but lower than the mouse receptor. The rank order of potency of a series of GnRH analogues for binding and inositol phosphate stimulation in transfected COS-1 cells was identical to that of the receptor characterized in sheep pituitary gonadotropes. Northern blot analysis identified four transcripts sized 5.4 kb, 3.6 kb, 2.3 kb and 1.3 kb in sheep pituitaries which were upregulated by castration.
Molecular and Cellular Endocrinology, 1994
Molecular and Cellular Endocrinology, 1993
Molecular Endocrinology, 2002
GnRH regulates the reproductive system through cognate G protein-coupled receptors in vertebrates... more GnRH regulates the reproductive system through cognate G protein-coupled receptors in vertebrates. Certain GnRH analogs that are antagonists at mammalian receptors behave as agonists at Xenopus laevis and chicken receptors. This phenomenon provides the opportunity to elucidate interactions and the mechanism underlying receptor activation. A D-Lys(iPr) in position 6 of the mammalian GnRH receptor antagonist is required for this agonist activity (inositol phosphate production) in the chicken and X. laevis GnRH receptors. Chimeric receptors, in which extracellular loop domains of the human GnRH receptor were substituted with the equivalent domains of the X. laevis GnRH receptor, identified extracellular loop 2 as the determinant for agonist activity of one of the mammalian antagonists: antagonist 135-18. Sitedirected mutagenesis of nine nonconserved residues in the C-terminal domain of extracellular loop 2 of the human GnRH receptor showed that a minimum of two mutations (Val 5.24(197) Ala and Trp 5.32(205) His) is needed in this region for agonist activity of antagonist 135-18. Agonist activity of antagonist 135-18 was markedly decreased by low pH (<7.0) compared with GnRH agonists. These findings indicate that D-Lys(iPr) 6 forms a chargesupported hydrogen bond with His 5.32(205) to stabilize the receptor in the active conformation. This discovery highlights the importance of EL-2 in ligand binding and receptor activation in G proteincoupled receptors. (Molecular Endocrinology 16: 1079-1088, 2002) Abbreviations: CHR, Chimeric receptor; GPCR: G proteincoupled receptor; EL, extracellular loop domain; IP, inositol phosphate; TM, transmembrane domain.
Endocrinology, 2007
Multiple GnRH receptors are known to exist in nonmammalian species, but it is uncertain which rec... more Multiple GnRH receptors are known to exist in nonmammalian species, but it is uncertain which receptor type regulates reproduction via the hypothalamic-pituitary-gonadal axis. The teleost fish, Astatotilapia burtoni, is useful for identifying the GnRH receptor responsible for reproduction, because only territorial males reproduce. We have cloned a second GnRH receptor in A. burtoni, GnRH-R1 SHS (SHS is a peptide motif in extracellular loop 3), which is up-regulated in pituitaries of territorial males. We have shown that GnRH-R1 SHS is expressed in many tissues and specifically colocalizes with LH in the pituitary. In A. burtoni brain, mRNA levels of both GnRH-R1 SHS and a previously identified receptor, GnRH-R2 PEY , are highly correlated with mRNA levels of all three GnRH ligands. Despite its likely role in reproduction, we found that GnRH-R1 SHS has the highest affinity for GnRH2 in vitro and low responsivity to GnRH1. Our phylogenetic anal-
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Papers by Colleen Flanagan