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Review
. 2020 Sep 18;25(18):4272.
doi: 10.3390/molecules25184272.

A Paradigm for Peptide Hormone-GPCR Analyses

Fred Naider  1 , Jeffrey M Becker  2
Affiliations
Review

A Paradigm for Peptide Hormone-GPCR Analyses

Fred Naider et al. Molecules. .

Abstract

Work from our laboratories over the last 35 years that has focused on Ste2p, a G protein-coupled receptor (GPCR), and its tridecapeptide ligand α-factor is reviewed. Our work utilized the yeast Saccharomyces cerevisiae as a model system for understanding peptide-GPCR interactions. It explored the structure and function of synthetic α-factor analogs and biosynthetic receptor domains, as well as designed mutations of Ste2p. The results and conclusions are described using the nuclear magnetic resonance interrogation of synthetic Ste2p transmembrane domains (TMs), the fluorescence interrogation of agonist and antagonist binding, the biochemical crosslinking of peptide analogs to Ste2p, and the phenotypes of receptor mutants. We identified the ligand-binding domain in Ste2p, the functional assemblies of TMs, unexpected and interesting ligand analogs; gained insights into the bound α-factor structure; and unraveled the function and structures of various Ste2p domains, including the N-terminus, TMs, loops connecting the TMs, and the C-terminus. Our studies showed interactions between specific residues of Ste2p in an active state, but not resting state, and the effect of ligand activation on the dimerization of Ste2p. We show that, using a battery of different biochemical and genetic approaches, deep insight can be gained into the structure and conformational dynamics of GPCR-peptide interactions in the absence of a crystal structure.

Keywords: G protein-coupled receptors; Saccharomyces cerevisiae; chemical crosslinking; fluorescence screening; nuclear magnetic resonance; peptide analogs; peptide pheromone; photoactivated crosslinking; receptor mutation; receptor-ligand interaction.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
A diagrammatic representation of the pheromones (α-factor and a-factor) mediating the sexual conjugation of Saccharomyces cerevisiae haploid cells of opposite mating types (MATα and MATa). MATα and MATa cells secrete their cognate pheromones to prepare their opposite mating type for mating (conjugation), resulting in a diploid cell. Ste2p and Ste3p are the membrane-bound receptors on MATa and MATα cells, respectively, that recognize their bound pheromones to arrest the recipient cell in the G1 phase of the cell cycle in preparation for conjugation. Pheromones and hormones perform physiologically equivalent functions; pheromones act between individuals of the same species, whereas hormones are produced and act within an individual.
Figure 2
Figure 2
Snake diagram of Ste2p. A 2D representation of Ste2p showing the N-terminus, seven-transmembrane TM bundle, C-terminus (CT), and three extracellular (EL) and three intracellular (IL) loops that connect TMs to each other. The TMs are embedded in the plasma membrane (PM), with the N-terminus facing extracellularly and the C-terminus facing intracellularly. The presence of an 8th helix is indicated, as will be discussed later in the review.
Figure 3
Figure 3
Biologically active conformation of α-factor. The tridecapeptide is flexible around residues 7–10, which we believe form a β-turn-like loop. After the three C-terminal residues of the peptide bind to Ste2p, the loop region allows the “bending” of α-factor to form a conformation in which the three N-terminal residues productively interact with Ste2p to initiate signal transduction.
Figure 4
Figure 4
Biased agonism. This diagram shows two peptides, one the naturally occurring α-factor, a normal agonist (NA), and the other a biased agonist (BA) that preferentially initiates different signaling pathways (morphogenesis or agglutination). The BA initiates agglutination at a much lower concentration than that of NA, whereas the NA initiates both similarly [44].
Figure 5
Figure 5
Structures of tyrosine (Tyr), 3,4-dihydoxyphenylanine (DOPA), tryptophan (Trp), and 4-benzoylphenylalanine (BPA).
Figure 6
Figure 6
Endocytosis of [K7(NBD),Nle12]α-factor detected with fluorescence microscopy. Cells expressing full-length receptors (left panels) or truncated receptors (right panels) from multicopy plasmids were incubated with the fluorescent ligand at 30 °C for (a,e) 1, (b,f) 20, (c,g) 60, and (d,h) 120 min. The ranges of image intensities in panels (e–h) are approximately 2-fold greater than those for panels (a–d), indicative of the stronger fluorescence from the truncated receptors. Taken with permission from [70].
Figure 7
Figure 7
Model for the binding and activation of normal and mutant α-factor receptors by different ligands. Ligand-interacting surfaces on the receptor are indicated by cross-hatching. Site I interacts with the C-termini of ligands and is likely to consist of the extracellular end of the first transmembrane segment. Site 2 interacts with the N-terminus of ligands and most likely consists of the extracellular end of the sixth transmembrane segment and the third extracellular loop of the receptor. The conformational equilibrium between the inactive and activated states of the receptor is represented as a change in the distance between Site 1 and Site 2. (a) In the absence of ligand, the receptor favors the inactivated state with a large separation between ligand contact sites. (b) Binding of the agonist to normal receptors favors the activated state. (c) Mutations affecting the emission spectrum of bound agonist change the environment of the fluorophore (red shift) without altering the receptor ligand binding or, generally, activation. (d) Binding of ligands that act as antagonists toward normal receptors (labeled antagonist) does not alter the conformational equilibrium of the receptor. Activation-specific interactions with the N-terminus of the ligand are blocked by the d-Tyr3 substitution (orange circle), leaving the NBD group attached at Lys7 exposed to the solvent, and the C-terminal region of the antagonist (which is identical to the corresponding region of agonist) maintains a high-affinity interaction with the receptor. Adapted with permission from [71].
Figure 8
Figure 8
(A) Backbone representation of the ensemble of the 20 lowest energy conformers of Ste2p (G31-T110) superimposed over backbone atoms in the TM1-TM2 region comprising residues 39–103. Observed long-range NOE contacts are highlighted in red. (B) A single conformer from the ensemble additionally displaying the side chains. (C) Structure of a single conformer—view from the side of the membrane interior. (D) The same as C, but viewed from the cytoplasmic side. Taken with permission from [121].
Figure 9
Figure 9
Binding of α-factor to Ste2p. Glutamine (Q10) of the C-terminus of α-factor binds to residues Ser47 and Thr48 at the interface of the receptor’s N-terminus and the extracellular face of TM1 and tyrosine (Y13) binds to Arg58 and/or Cys59 in TM1. The bending of the pheromone around the loop region facilitates the interaction between the tryptophan (W1) at the N-terminus of α-factor and residues Y266 and K269 in TM6 and EL3. The N-terminus is shown interacting with EL1. α-Factor residues are indicated in black (not all the residues of the pheromone are shown in the figure), Ste2p residues are shown in blue, Ste2p loops and termini are in red, and transmembrane domains are in gray.
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References

    1. Hauser M., Narita V., Donhardt A.M., Naider F., Becker J.M. Multiplicity and regulation of genes encoding peptide transporters in Saccharomyces cerevisiae. Mol. Membr. Biol. 2001;18:105–112. doi: 10.1080/09687680010029374. - DOI - PubMed
    1. Alvaro C.G., Thorner J. Heterotrimeric G Protein-coupled Receptor Signaling in Yeast Mating Pheromone Response. J. Biol. Chem. 2016;291:7788–7795. doi: 10.1074/jbc.R116.714980. - DOI - PMC - PubMed
    1. Bucking-Throm E., Duntze W., Hartwell L.H., Manney T.R. Reversible arrest of haploid yeast cells in the initiation of DNA synthesis by a diffusible sex factor. Exp. Cell Res. 1973;76:99–110. doi: 10.1016/0014-4827(73)90424-2. - DOI - PubMed
    1. Stotzler D., Duntze W. Isolation and characterization of four related peptides exhibiting alpha factor activity from Saccharomyces cerevisiae. Eur. J. Biochem. 1976;65:257–262. doi: 10.1111/j.1432-1033.1976.tb10412.x. - DOI - PubMed
    1. Masui Y., Chino N., Sakakibara S., Tanaka T., Murakami T., Kita H. Synthesis of the mating factor of Saccharomyces cerevisiae and its truncated peptides: The structure-activity relationship. Biochem. Biophys. Res. Commun. 1977;78:534–538. doi: 10.1016/0006-291X(77)90211-X. - DOI - PubMed

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