Identification of Ligand Binding Regions of the Saccharomyces cerevisiae -Factor Pheromone Receptor by Photoaffinity Cross-Li
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- 作者：Cagdas D. Son ; Hasmik Sargsyan ; Fred Naider ; and Jeffrey M. Becker
- 刊名：Biochemistry
- 出版年：2004
- 出版时间：October 19, 2004
- 年：2004
- 卷：43
- 期：41
- 页码：13193 - 13203
- 全文大小：157K
- 年卷期：v.43,no.41(October 19, 2004)
- ISSN：1520-4995
文摘
Analogues of 
-factor, Saccharomyces cerevisiae tridecapeptide mating pheromone (H-Trp-His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr-OH), containing p-benzoylphenylalanine (Bpa), aphotoactivatable group, and biotin as a tag, were synthesized using solid-phase methodologies on ap-benzyloxybenzyl alcohol polystyrene resin. Bpa was inserted at positions 1, 3, 5, 8, and 13 of -factorto generate a set of cross-linkable analogues spanning the pheromone. The biological activity (growtharrest assay) and binding affinities of all analogues for the -factor receptor (Ste2p) were determined.Two of the analogues that were tested, Bpa1 and Bpa5, showed 3-4-fold lower affinity than the -factor,whereas Bpa3 and Bpa13 had 7-12-fold lower affinities. Bpa8 competed poorly with [3H]--factor forSte2p. All of the analogues tested except Bpa8 had detectable halos in the growth arrest assay, indicatingthat these analogues are -factor agonists. Cross-linking studies demonstrated that [Bpa1]--factor, [Bpa3]--factor, [Bpa5]--factor, and [Bpa13]--factor were cross-linked to Ste2p; the biotin tag on the pheromonewas detected by a NeutrAvidin-HRP conjugate on Western blots. Digestion of Bpa1, Bpa3, and Bpa13cross-linked receptors with chemical and enzymatic reagents suggested that the N-terminus of thepheromone interacts with a binding domain consisting of residues from the extracellular ends of TM5-TM7 and portions of EL2 and EL3 close to these TMs and that there is a direct interaction between theposition 13 side chain and a region of Ste2p (F55-R58) at the extracellular end of TM1. The resultsfurther define the sites of interaction between Ste2p and the -factor, allowing refinement of a model forthe pheromone bound to its receptor.
-factor, Saccharomyces cerevisiae tridecapeptide mating pheromone (H-Trp-His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr-OH), containing p-benzoylphenylalanine (Bpa), aphotoactivatable group, and biotin as a tag, were synthesized using solid-phase methodologies on ap-benzyloxybenzyl alcohol polystyrene resin. Bpa was inserted at positions 1, 3, 5, 8, and 13 of -factorto generate a set of cross-linkable analogues spanning the pheromone. The biological activity (growtharrest assay) and binding affinities of all analogues for the -factor receptor (Ste2p) were determined.Two of the analogues that were tested, Bpa1 and Bpa5, showed 3-4-fold lower affinity than the -factor,whereas Bpa3 and Bpa13 had 7-12-fold lower affinities. Bpa8 competed poorly with [3H]--factor forSte2p. All of the analogues tested except Bpa8 had detectable halos in the growth arrest assay, indicatingthat these analogues are -factor agonists. Cross-linking studies demonstrated that [Bpa1]--factor, [Bpa3]--factor, [Bpa5]--factor, and [Bpa13]--factor were cross-linked to Ste2p; the biotin tag on the pheromonewas detected by a NeutrAvidin-HRP conjugate on Western blots. Digestion of Bpa1, Bpa3, and Bpa13cross-linked receptors with chemical and enzymatic reagents suggested that the N-terminus of thepheromone interacts with a binding domain consisting of residues from the extracellular ends of TM5-TM7 and portions of EL2 and EL3 close to these TMs and that there is a direct interaction between theposition 13 side chain and a region of Ste2p (F55-R58) at the extracellular end of TM1. The resultsfurther define the sites of interaction between Ste2p and the -factor, allowing refinement of a model forthe pheromone bound to its receptor.