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Research ArticleArticle

Secretin Amino-Terminal Structure-Activity Relationships and Complementary Mutagenesis at the Site of Docking to the Secretin Receptor

Juliana E. Milburn, Kaleeckal G. Harikumar, Sarah J. Piper, Sweta Raval, Arthur Christopoulos, Denise Wootten, Patrick M. Sexton and Laurence J. Miller
Molecular Pharmacology June 2022, 101 (6) 400-407; DOI: https://doi.org/10.1124/molpharm.122.000502
Juliana E. Milburn
Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Scottsdale, Arizona (J.E.M., K.G.H., S.R., L.J.M.) and Drug Discovery Biology and Australian Research Council Centre for Cryo-Electron Microscopy of Membrane Proteins, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Australia (S.J.P., A.C., D.W., P.M.S.)
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Kaleeckal G. Harikumar
Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Scottsdale, Arizona (J.E.M., K.G.H., S.R., L.J.M.) and Drug Discovery Biology and Australian Research Council Centre for Cryo-Electron Microscopy of Membrane Proteins, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Australia (S.J.P., A.C., D.W., P.M.S.)
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Sarah J. Piper
Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Scottsdale, Arizona (J.E.M., K.G.H., S.R., L.J.M.) and Drug Discovery Biology and Australian Research Council Centre for Cryo-Electron Microscopy of Membrane Proteins, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Australia (S.J.P., A.C., D.W., P.M.S.)
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Sweta Raval
Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Scottsdale, Arizona (J.E.M., K.G.H., S.R., L.J.M.) and Drug Discovery Biology and Australian Research Council Centre for Cryo-Electron Microscopy of Membrane Proteins, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Australia (S.J.P., A.C., D.W., P.M.S.)
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Arthur Christopoulos
Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Scottsdale, Arizona (J.E.M., K.G.H., S.R., L.J.M.) and Drug Discovery Biology and Australian Research Council Centre for Cryo-Electron Microscopy of Membrane Proteins, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Australia (S.J.P., A.C., D.W., P.M.S.)
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Denise Wootten
Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Scottsdale, Arizona (J.E.M., K.G.H., S.R., L.J.M.) and Drug Discovery Biology and Australian Research Council Centre for Cryo-Electron Microscopy of Membrane Proteins, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Australia (S.J.P., A.C., D.W., P.M.S.)
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Patrick M. Sexton
Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Scottsdale, Arizona (J.E.M., K.G.H., S.R., L.J.M.) and Drug Discovery Biology and Australian Research Council Centre for Cryo-Electron Microscopy of Membrane Proteins, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Australia (S.J.P., A.C., D.W., P.M.S.)
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Laurence J. Miller
Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Scottsdale, Arizona (J.E.M., K.G.H., S.R., L.J.M.) and Drug Discovery Biology and Australian Research Council Centre for Cryo-Electron Microscopy of Membrane Proteins, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Australia (S.J.P., A.C., D.W., P.M.S.)
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Abstract

Class B1 G protein–coupled receptors are activated by peptides, with amino-terminal regions critical for biologic activity. Although high resolution structures exist, understanding of key features of the peptide activation domain that drive signaling is limited. In the secretin receptor (SecR) structure, interactions are observed between peptide residues His1 and Ser2 and seventh transmembrane segment (TM7) receptor residue E373. We interrogated these interactions using systematic structure-activity analysis of peptide and receptor. His1 was critical for binding and cAMP responses, but its orientation was not critical, and substitution could independently modify affinity and efficacy. Ser2 was also critical, with all substitutions reducing peptide affinity and functional responses proportionally. Mutation of E373 to conserved acidic Asp (E373D), uncharged polar Gln (E373Q), or charge-reversed basic Arg (E373R) did not alter receptor expression, with all exhibiting secretin-dependent cAMP accumulation. All position 373 mutants displayed reduced binding affinities and cAMP potencies for many peptide analogs, although relative effects of position 1 peptides were similar whereas position 2 peptides exhibited substantial differences. The peptide including basic Lys in position 2 was active at SecR having acidic Glu in position 373 and at E373D while exhibiting minimal activity at those receptors in which an acidic residue is absent in this position (E373Q and E373R). In contrast, the peptide including acidic Glu in position 2 was equipotent with secretin at E373R while being much less potent than secretin at wild-type SecR and E373D. These data support functional importance of a charge-charge interaction between the amino-terminal region of secretin and the top of TM7.

SIGNIFICANCE STATEMENT This work refines our molecular understanding of the activation mechanisms of class B1 G protein–coupled receptors. The amino-terminal region of secretin interacts with the seventh transmembrane segment of its receptor with structural specificity and with a charge-charge interaction helping to drive functional activation.

Footnotes

    • Received February 9, 2022.
    • Accepted March 22, 2022.
  • This work was supported by a grant from National Institutes of Health National Institute of General Medical Sciences [Grant R01-GM132095]. P.M.S. is a Senior Principal Research Fellow of the National Health and Medical Research Council of Australia [ID: 1154434]. D.W. is a Senior Research Fellow of the National Health and Medical Research Council of Australia [ID: 1155302].

  • P.M.S, A.C., and D.W. are shareholders in Septerna Inc. No author has an actual or perceived conflict of interest with the contents of this article.

  • https://doi.org/10.1124/molpharm.122.000502.

  • Copyright © 2022 by The American Society for Pharmacology and Experimental Therapeutics
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Molecular Pharmacology: 101 (6)
Molecular Pharmacology
Vol. 101, Issue 6
1 Jun 2022
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Research ArticleArticle

Secretin Amino-Terminal Structure-Activity Relationships and Complementary Mutagenesis at the Site of Docking to the Secretin Receptor

Juliana E. Milburn, Kaleeckal G. Harikumar, Sarah J. Piper, Sweta Raval, Arthur Christopoulos, Denise Wootten, Patrick M. Sexton and Laurence J. Miller
Molecular Pharmacology June 1, 2022, 101 (6) 400-407; DOI: https://doi.org/10.1124/molpharm.122.000502

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Research ArticleArticle

Secretin Amino-Terminal Structure-Activity Relationships and Complementary Mutagenesis at the Site of Docking to the Secretin Receptor

Juliana E. Milburn, Kaleeckal G. Harikumar, Sarah J. Piper, Sweta Raval, Arthur Christopoulos, Denise Wootten, Patrick M. Sexton and Laurence J. Miller
Molecular Pharmacology June 1, 2022, 101 (6) 400-407; DOI: https://doi.org/10.1124/molpharm.122.000502
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