Kelch Repeat Protein Interacts with the Yeast Gα Subunit Gpa2p at a Site That Couples Receptor Binding to Guanine Nucleotide Exchange

Thiruvur Niranjan,Xuedong Guo,Jacob Victor,Ailan Lu,Jeanne P Hirsch

doi:10.1074/jbc.m702595200

Thiruvur Niranjan, Xuedong Guo + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m702595200

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Abstract

The kelch repeat-containing proteins Krh1p and Krh2p are negative regulators of the Gpa2p signaling pathway that directly interact with the G protein alpha-subunit Gpa2p in the yeast Saccharomyces cerevisiae. A screen was carried out to identify Gpa2p variants that are defective in their ability to bind Krh1p but retain the ability to bind another Gpa2p-interacting protein, Ime2p. This screen identified amino acids Gln-419 and Asn-425 as being important for the interaction between Gpa2p and Krh1p. Gpa2p variants with changes at these positions are defective for Krh1p binding in vivo. Cells containing these forms of Gpa2p display decreased heat shock resistance and increased expression of a gene required for pseudohyphal growth. These findings indicate that the substitutions at positions 419 and 425 confer a degree of constitutive activity to the Gpa2p alpha-subunit. Residues Gln-419 and Asn-425 are located in the beta6-alpha5 loop and alpha5 helix of Gpa2p, which is the region that couples receptor binding to guanine nucleotide exchange. The results suggest that binding of Gpa2p to Krh1p does not resemble the binding of Galpha subunits to either Gbeta subunits or effectors, but it instead represents a novel type of functional interaction.

Highlights

AUGUST 17, 2007 VOLUME 282 NUMBER 33 resistance [3, 6, 7]. gpa2⌬ mutants display an increase in replicative life span, which is the number of mitotic divisions completed by a cell prior to senescence (8 –10)
The importance of the ␤6-␣5 region was demonstrated recently by the finding that the coupling of receptor binding to guanine nucleotide exchange requires movement of the ␣5 helix of the G␣ subunit [33]
In our study, binding of Gpa2p to Krh1p was shown to be affected by altering amino acids in the ␣5 helix and the ␤6-␣5 loop, a region that is critical for transmitting the structural change that couples receptor binding to GDP release

Summary

Introduction

AUGUST 17, 2007 VOLUME 282 NUMBER 33 resistance [3, 6, 7]. gpa2⌬ mutants display an increase in replicative life span, which is the number of mitotic divisions completed by a cell prior to senescence (8 –10). The Gpr1p receptor is required for pseudohyphal and invasive growth, and this requirement can be overcome by constitutive activation of Gpa2p, as would be expected for a G protein that acts downstream of its coupled receptor [13, 14]. At least one component of Gpa2p signaling appears to involve stimulation of adenylyl cyclase by Gpa2p, resulting in production of cAMP and activation of protein kinase A (PKA).. Recent evidence indicates that adenylyl cyclase binds to Gpa2p in a manner that is highly specific for the GTP-bound form of the ␣-subunit [5]. Stimulation of adenylyl cyclase activates the cAMP-dependent kinase PKA, and genetic evidence indicates that Gpa2p functions as a positive regulator of PKA signaling. Deletion of TPK2, which encodes the PKA catalytic subunit required for filamentous growth, eliminates the effect of constitutively

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