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In Vitro Mutational Analysis of the β2 Adrenergic Receptor, an In Vivo Surrogate Odorant Receptor

Sophie Jamet1, Jaclyn Bubnell1, Patrick Pfister2

  • 1Department of Biological Sciences, Hunter College, CUNY, New York, New York, United States of America.

Plos One
|October 30, 2015
PubMed

Insights

Investigating G-protein coupled receptor (GPCR) trafficking, this study reveals that specific mutations in the mouse β2-adrenergic-receptor (mβ2AR) impact plasma membrane localization and signaling. Carboxy-terminal mutations are critical for G-protein coupling and receptor function.

Area of Science:

  • Molecular and Cellular Biology
  • Neuroscience
  • Biochemistry

Background:

  • Many G-protein coupled receptors (GPCRs), including odorant receptors (ORs), exhibit poor plasma membrane trafficking in heterologous cells, hindering their characterization.
  • The mouse β2-adrenergic-receptor (mβ2AR), a surrogate GPCR, demonstrates robust plasma membrane localization, making it a valuable model for studying GPCR trafficking and function.
  • Understanding GPCR trafficking is crucial for developing targeted therapeutics and advancing fields like olfactory axon guidance.

Purpose of the Study:

  • To characterize in vitro mutants of the mβ2AR for their potential application in olfactory axon guidance studies.
  • To investigate the roles of the amino-terminal (Nt) and carboxy-terminal (Ct) domains of mβ2AR in plasma membrane localization and ligand-induced signaling.
  • To identify specific mutations affecting mβ2AR trafficking and signaling to inform models of GPCR folding and transport.

Main Methods:

  • Extensive mutational analysis of mβ2AR using a Green Fluorescent Protein-tagged construct (mβ2AR::GFP) to assess plasma membrane localization.
  • Determination of half-maximal effective concentrations (EC50) and maximal responses to isoprenaline to evaluate ligand-initiated signal transduction.
  • Analysis of specific mβ2AR mutants (RDY, E268A, C327R) and domain-swapped constructs (mβ2AR with M71 OR Nt/Ct) for trafficking and signaling capacity.

Main Results:

  • Removal of Nt N-glycosylation sites and Ct palmitoylation sites did not impede mβ2AR plasma membrane localization.
  • Replacing both Nt and Ct of mβ2AR with those from M71 OR significantly impaired plasma membrane trafficking.
  • A deletion of the Ct abolished mβ2AR activity, but a specific mutation (Y to A in NPxxY motif) rescued trafficking, suggesting a retention signal mechanism.
  • Ct mutations uniquely reduced mβ2AR EC50 values, highlighting their critical role in G-protein coupling.
  • Trafficking and isoprenaline response of certain mutants (RDY, E268A, C327R) were decorrelated, indicating distinct regulatory mechanisms.

Conclusions:

  • The carboxy-terminal domain of mβ2AR plays a crucial role in G-protein coupling and overall receptor activity.
  • Specific residues at the end of transmembrane domain 7 may function as a retention signal when unmasked.
  • An interaction between the amino-terminal and carboxy-terminal domains is likely essential for proper GPCR folding and/or transport.

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