Photoaffinity labeling of pituitary GnRH receptors: significance of the position of photolabel on the ligand

Nikolics, Károly; Szonyi, Eva; Ramachandran, J.

doi:10.1021/bi00405a006

Biochemistry

1988

DOI: 10.1021/bi00405a006

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Photoaffinity labeling of pituitary GnRH receptors: significance of the position of photolabel on the ligand

Károly Nikolics¹,

Eva Szonyi²,

J. Ramachandran³

Abstract: Photoreactive derivatives of GnRH and its analogues were prepared by incorporation of the 2-nitro-4(5)-azidophenylsulfenyl [2,4(5)-NAPS] group into amino acid residues at positions 1, 3, 6, or 8 of the decapeptide sequence. The modification of Trp3 by the 2,4-NAPS group led to a complete loss of the luteinizing hormone (LH) releasing as well as LH-release-inhibiting activity of the peptide. The [D-Lys(2,4-NAPS)]6 analogue was a very potent agonist that, after covalent attachment by photoaffinity labeling, caus… Show more

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Cited by 9 publications

(1 citation statement)

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“…LHRH receptors. The central LHRH fragment is probably not involved directly in receptor binding, since even an attachment of a macromolecule like polyglutamic acid or polyethylene glycol to LHRH via the side chain in position 6 does not influence significantly its agonistic potency (see ref 11. and references therein).…”

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confidence: 99%

Conformation‐function relationships in LHRH analogs

Nikiforovich

Marshall

1993

International Journal of Peptide and Protein Research

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Systematic energy calculations were performed for a series of LHRH analogs including five agonists with substitutions of d‐ or N‐Me‐amino acid residues in positions 4, 6 and 7, and five antagonists with substitutions of d‐, N‐Me‐ or α‐Me‐amino acid residues in positions 1, 2, 3, 6, 7 and 10, as well as a bicyclic LHRH antagonist. The geometrical shapes of the calculated low‐energy backbone structures for each compound were compared to those of LHRH itself. It appeared that the β‐II′ turn at the Tyr5‐Gly6‐Leu7‐Arg8 central tetrapeptide is the common structure for all LHRH agonists considered. LHRH antagonists also possess a common chain reversal in the central tetrapeptide, but it is different from that for LHRH agonists. The LHRH agonists share a similar low‐energy conformer at the level of the entire peptide backbone. A characteristic feature of this conformer is a ‘surface’ formed by a ‘polygon’ with hydrophobic moieties of pGlu1, Trp3, Tyr5, Leu7 and Pro9 in the corners and with the side chain of the His2 residue in the middle, the latter being crucial for a manifestation of LHRH agonistic activity. Since the N‐terminal tripeptide of LHRH presumably participates in a direct interaction with specific receptors, it is legitimate to suggest that the β‐II′ turn in the central tetrapeptide maintains the proper spatial arrangement of the N‐terminal tripeptide. On the other hand, LHRH antagonists considered in this study were shown to possess low‐energy structures, with the spatial arrangement of the residues in the N‐terminal tripeptide similar to that of agonists. This would suggest a new approach to the design of LHRH antagonists, namely by stabilizing this specific arrangement, rather than the β‐II′ turn in the central tetrapeptide.

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mentioning

confidence: 99%

Conformation‐function relationships in LHRH analogs

Nikiforovich

Marshall

1993

International Journal of Peptide and Protein Research

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The structural features of effective antagonists of the luteinizing hormone releasing hormone

et al. 1994

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The structure-activity data of 6 years on 395 analogs of the luteinizing hormone releasing hormone (LHRH) have been studied to determine effective substituents for the ten positions for maximal antiovulatory activity and minimal histamine release. The numbers of substituents studied in the ten positions are as follows: (41)(1)-(12)(2)-(12)(3)-(5)(4)-(47)(5)-(52)(6)-(16)(7)-(18)(8)-(4)(9)-(8)(10). In position 1, DNal and DQal were effective with the former being more frequently the better substituent. DpClPhe was uniquely effective in position 2. Positions 3 and 4 are very sensitive to change. D3Pal in position 3 and Ser in position 4 of LHRH were in the best antagonists. PicLys and cPzACAla were the most successful residues in position 5 with cPzACAla being the better substituent. Position 6 was the most flexible and many substituents were effective; particularly DPicLys. Leu(7) was most often present in the best antagonists. In position 8, Arg was effective for both antiovulatory activity and histamine release; ILys was effective for potency and lesser histamine release. Pro(9) of LHRH was retained. DAlaNH2 (10) was in the best antagonists.

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Incorporation of an additional glycosylation site enhances expression of functional human gonadotropin-releasing hormone receptor

Davidson

Flanagan

Davies

et al. 1996

Endocr

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Mutation ofN-glycosylation sites in the mouse gonadotropin-releasing hormone receptor was previously shown to impair its expression in COS-1 cells. We therefore investigated the effects of adding an extra glycosylation site to the human gonadotropin-releasing hormone receptor, as a means for increasing its expression. Covalent labeling of the mutant receptor expressed in COS-1 cells with a gonadotropin-releasing hormone (GnRH) photoreactive analog demonstrated a shift in apparent molecular weight, indicating that the new site was in fact glycosylated. The receptor with extra glycosylation site displayed normal binding affinities for agonists buserelin and [D: -Ala(6)-Pro(9)-NHEt]-GnRH, and the antagonist antide, and a slightly increased affinity for GnRH. Receptor number was increased by 1.7-fold in membrane preparations from cells expressing the mutant receptor, compared with wild-type. Photoaffinity labeling of cell-surface receptors in intact cells demonstrated a 1.8-fold increase in binding sites on the cell surface. The GnRH receptor (GnRHR) with extra glycosylation site conferred a markedly enhanced signaling response to agonist. Dose-response curves for GnRH-stimulated inositol phosphate production were left-shifted by an average of 4.4-fold, and maximal inositol phosphate responses were increased by 1.2 fold, in cells transfected with mutant compared with wild-type receptor, indicating that the increase in binding sites represented functional receptors. These results demonstrate that addition of an extra glycosylation site enhances expression of the human GnRHR, a strategy that may be applicable to other cell-surface receptors.

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Photoaffinity labeling of pituitary GnRH receptors: significance of the position of photolabel on the ligand

Cited by 9 publications

References 39 publications

Conformation‐function relationships in LHRH analogs

Conformation‐function relationships in LHRH analogs

The structural features of effective antagonists of the luteinizing hormone releasing hormone

Incorporation of an additional glycosylation site enhances expression of functional human gonadotropin-releasing hormone receptor

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