Topogenesis and cell surface trafficking of GPR34 are facilitated by positive-inside rule that effects through a tri-basic motif in the first intracellular loop.

Authors:
Haruki Hasegawa, PhD
Haruki Hasegawa, PhD
Amgen Inc.
Principal Scientist
Protein trafficking Protein biosynthesis
South San Francisco, CA | United States
Neha Patel
Neha Patel
Department of Otolaryngology
St. Louis | United States
Elham Ettehadieh
Elham Ettehadieh
University of British Columbia
Canada
Peng Li
Peng Li
State Key Laboratory of Quality Research in Chinese Medicine
China

Biochim Biophys Acta 2016 Jul 14;1863(7 Pt A):1534-51. Epub 2016 Apr 14.

Department of Therapeutic Discovery, Amgen Inc., South San Francisco, CA 94080, USA.

Protein folding, topogenesis and intracellular targeting of G protein-coupled receptors (GPCRs) must be precisely coordinated to ensure correct receptor localization. To elucidate how different steps of GPCR biosynthesis work together, we investigated the process of membrane topology determination and how it relates to the acquisition of cell surface trafficking competence in human GPR34. By monitoring a fused FLAG-tag and a conformation-sensitive native epitope during the expression of GPR34 mutant panel, a tri-basic motif in the first intracellular loop was identified as the key topogenic signal that dictates the orientation of transmembrane domain-1 (TM1). Charge disruption of the motif perturbed topogenic processes and resulted in the conformational epitope loss, post-translational processing alteration, and trafficking arrest in the Golgi. The placement of a cleavable N-terminal signal sequence as a surrogate topogenic determinant overcame the effects of tri-basic motif mutations and rectified the TM1 orientation; thereby restored the conformational epitope, post-translational modifications, and cell surface trafficking altogether. Progressive N-tail truncation and site-directed mutagenesis revealed that a proline-rich segment of the N-tail and all four cysteines individually located in the four separate extracellular regions must simultaneously reside in the ER lumen to muster the conformational epitope. Oxidation of all four cysteines was necessary for the epitope formation, but the cysteine residues themselves were not required for the trafficking event. The underlying biochemical properties of the conformational epitope was therefore the key to understand mechanistic processes propelled by positive-inside rule that simultaneously regulate the topogenesis and intracellular trafficking of GPR34.

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http://dx.doi.org/10.1016/j.bbamcr.2016.04.010DOI Listing

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July 2016
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