Analysis of Hormone-Receptor Interaction Sites Using Synthetic Peptides: Receptor Binding Regions of the α-Subunit of Human Choriogonadotropin

Dean E. Morbeck, M. Cristine Charlesworth, Dana Kim Reed, Daniel J Mc Cormick

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

Synthetic peptides are valuable tools for determining sites of interaction between hormones and their receptors. We have learned much about linear receptor binding regions of the glycoprotein hormone human choriogonadotropin (hCG) using synthetic peptides corresponding to its primary sequence. Of paramount importance in any study using synthetic peptides as a tool to investigate protein structure are an efficient means of peptide purification and a definitive measure of peptide purity and composition. Purification is easily achieved for all but the most hydrophobic peptides using preparative reverse-phase HPLC. Of the methods available for analysis of peptide purity, mass spectrometry is perhaps the most useful and often most rapid approach. Other essential components of studies involving synthetic peptides and hormone binding are reproducible hormone labeling, receptor preparations, and bioassays. The ability of peptides to compete with hCG for binding to specific receptors is tested in radioreceptor binding assays and bioassays with transformed Leydig cell lines. These cells express the lutropin receptor that is coupled to a measurable endpoint such as cAMP or steroid production. The conditions for these assays are optimized for rapid and accurate measurement of peptide activity. Since the three-dimensional structure of hCG is not known, a systematic approach to the identification of potential receptor binding sites is advocated. First, a comprehensive analysis using synthetic overlapping peptides of uniform length that span the entire sequence of the α-subunit is employed. This approach is an effective means for surveying the entire subunit for receptor binding sites. Next, the boundaries of the active regions are delimited by a series of nested peptides sequentially shortened in length. The importance of each residue within the delimited binding regions is then studied using a series of peptides containing single alanine substitutions. Finally, modifications to enhance activity of the synthetic peptides are further made on the basis of data from alanine substitution studies, circular dichroic analysis, and molecular modeling. These studies provide valuable information to aid in the design of synthetic hormone analogs and for further analysis of the structure-function of hCG via site-directed mutagenesis.

Original languageEnglish (US)
Pages (from-to)191-200
Number of pages10
JournalMethods
Volume5
Issue number3
DOIs
StatePublished - Dec 1993

Fingerprint

Artificial Receptors
Peptide Receptors
Chorionic Gonadotropin
Hormones
Peptides
Bioassay
Biological Assay
Alanine
Purification
Assays
Substitution reactions
Binding Sites
LH Receptors
Radioligand Assay
Mutagenesis
Transformed Cell Line
Molecular modeling
Leydig Cells
Peptide Hormones
Surveying

ASJC Scopus subject areas

  • Molecular Biology

Cite this

Analysis of Hormone-Receptor Interaction Sites Using Synthetic Peptides : Receptor Binding Regions of the α-Subunit of Human Choriogonadotropin. / Morbeck, Dean E.; Charlesworth, M. Cristine; Reed, Dana Kim; Mc Cormick, Daniel J.

In: Methods, Vol. 5, No. 3, 12.1993, p. 191-200.

Research output: Contribution to journalArticle

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abstract = "Synthetic peptides are valuable tools for determining sites of interaction between hormones and their receptors. We have learned much about linear receptor binding regions of the glycoprotein hormone human choriogonadotropin (hCG) using synthetic peptides corresponding to its primary sequence. Of paramount importance in any study using synthetic peptides as a tool to investigate protein structure are an efficient means of peptide purification and a definitive measure of peptide purity and composition. Purification is easily achieved for all but the most hydrophobic peptides using preparative reverse-phase HPLC. Of the methods available for analysis of peptide purity, mass spectrometry is perhaps the most useful and often most rapid approach. Other essential components of studies involving synthetic peptides and hormone binding are reproducible hormone labeling, receptor preparations, and bioassays. The ability of peptides to compete with hCG for binding to specific receptors is tested in radioreceptor binding assays and bioassays with transformed Leydig cell lines. These cells express the lutropin receptor that is coupled to a measurable endpoint such as cAMP or steroid production. The conditions for these assays are optimized for rapid and accurate measurement of peptide activity. Since the three-dimensional structure of hCG is not known, a systematic approach to the identification of potential receptor binding sites is advocated. First, a comprehensive analysis using synthetic overlapping peptides of uniform length that span the entire sequence of the α-subunit is employed. This approach is an effective means for surveying the entire subunit for receptor binding sites. Next, the boundaries of the active regions are delimited by a series of nested peptides sequentially shortened in length. The importance of each residue within the delimited binding regions is then studied using a series of peptides containing single alanine substitutions. Finally, modifications to enhance activity of the synthetic peptides are further made on the basis of data from alanine substitution studies, circular dichroic analysis, and molecular modeling. These studies provide valuable information to aid in the design of synthetic hormone analogs and for further analysis of the structure-function of hCG via site-directed mutagenesis.",
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