Regulation of G protein-coupled cAMP receptor activation by a hydrophobic residue in transmembrane helix 3

Authors

  • Minghang Zhang,

    1. Department of Microbiology and Molecular Genetics, University of Texas Health Science Center Medical School, Houston, TX 77030, USA.
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    • Present addresses: Epoch Biolabs, 2825 Wilcrest Dr S160, Houston, TX 77042, USA;

  • Mousumi Goswami,

    1. Department of Microbiology and Molecular Genetics, University of Texas Health Science Center Medical School, Houston, TX 77030, USA.
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  • Satoshi Sawai,

    1. Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA.
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    • ERATO Complex Systems Biology Project, JST, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902, Japan;

  • Edward C. Cox,

    1. Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA.
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  • Dale Hereld

    Corresponding author
    1. Department of Microbiology and Molecular Genetics, University of Texas Health Science Center Medical School, Houston, TX 77030, USA.
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    • §

      Laboratory of Immunogenetics, NIAID/NIH, Rockville, MD 20852, USA.


*E-mail hereldd@niaid.nih.gov; Tel. (+1) 301 480 1430; Fax (+1) 301 480 2618.

Summary

cAR1, a G protein-coupled cAMP receptor, is essential for multicellular development of Dictyostelium. We previously identified a cAR1-Ile104 mutant that appeared to be constitutively activated based on its constitutive phosphorylation, elevated affinity for cAMP, and dominant-negative effects on development as well as specific cAR1 pathways that are subject to adaptation. To investigate how Ile104 might regulate cAR1 activation, we assessed the consequences of substituting it with all other amino acids. Constitutive phosphorylation of these Ile104 mutants varied broadly, suggesting that they are activated to varying extents, and was correlated with polarity of the substituting amino acid residue. Remarkably, all Ile104 substitutions, except for the most conservative, dramatically elevated the receptor's cAMP affinity. However, only a third of the mutants (those with the most polar substitutions) blocked development. These findings are consistent with a model in which polar Ile104 substitutions perturb the equilibrium between inactive and active cAR1 conformations in favour of the latter. Based on homology with rhodopsin, Ile104 is likely buried within inactive cAR1 and exposed to the cytoplasm upon activation. We propose that the hydrophobic effect normally promotes burial of Ile104 and hence cAR1 inactivation, while polar substitution of Ile104 mitigates this effect, resulting in activation.

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