| The beta1-adrenergic receptor (beta1AR) is a G protein coupled receptor (GPCR) critical to proper heart function. While beta 1AR function at the plasma membrane has been extensively studied, less is known about the biosynthesis, trafficking and modification of newly synthesized protein. We discovered a role for a peripheral Golgi protein, golgin-160, in the efficient trafficking of newly synthesized beta1AR. Cells depleted of golgin-160 by RNA interference had reduced surface levels of beta 1AR as measured by flow cytometry or radioactive ligand binding, and arrival at the plasma membrane of newly synthesized beta1AR was delayed.;The N-terminus of golgin-160 interacted with beta1AR expressed in cells, or by in vitro transcription and translation. The interaction site was mapped to a short region of golgin-160 (residues 140--257) and the third intracellular loop of beta1AR. This suggests that golgin-160 may influence the trafficking of beta1AR by direct binding, potentially by promoting a post-translational modification.;beta1AR was found to be palmitoylated at two distinct sites, on three cysteines (Cys392, Cys393 and Cys 414). While palmitoylation at the proximal site (Cys392,393 ) is common for GPCRs, palmitoylation at the distal site (Cys 414) has been described for only three other GPCRs. Additionally, immature beta1AR was only palmitoylated when the proximal site was available, suggesting that these sites are utilized by palmitoyl transferases localized to different parts of the secretory pathway. No defect in palmitoylation at either site was observed in cells depleted of golgin-160. Mutations to the palmitoylated cysteines, alone or in combination, did not alter the half-life or localization of beta1AR, and did not disrupt surface levels or dimerization. However, the coupling of beta1AR to second messenger signaling was found be impaired when either site was mutated, and nearly abolished when both sites were mutated.;These results have added to our knowledge of the physical structure and function of the clinically important beta1AR. Further studies examining the contributions of each palmitoylation site to the function of beta 1AR may reveal that these sites have independent functions. |
