Dissection of anterograde trafficking and sorting of GPCRs in primary neurons

G protein‐coupled receptors (GPCRs) play an important role in regulating the function of the nervous system. However, the molecular mechanisms underlying the targeting and sorting of GPCRs to the functional destination in neurons are not well understood. Here we investigated the role of small GTPase Rab43, via dominant negative inhibition and CRISPR‐Cas9‐mediated knockout, in the transport of several prototypic GPCRs to dendrites and spines and elucidated the possible mechanisms. We demonstrated that inhibition of Rab43 function significantly reduced the surface numbers of α2‐adrenergic receptors (ARs) and β2‐ARs, but not M3‐muscarinic receptors (MRs) in primary cultures of neurons as measured by live neuron radioligand binding. Confocal microscopy revealed that three receptors utilized similar structural determinants, including the highly conserved LL motif in the C‐termini, for their anterograde transport, and that Rab43 affected the expression of α2‐ARs, but not M3‐MRs and vesicular stomatitis virus glycoprotein, at both dendrites and spines. Interestingly, Rab43 only altered β2‐AR expression at spines, but not dendrites. We also found that Rab43 directly and activation‐dependently interacted with α2‐ARs, but not M3‐MRs, and the Rab43‐binding domain was located in the third intracellular loop of the receptor. The chimeric M3‐MRs containing the Rab43‐binding domain of α2‐ARs were subject to Rab43 regulation. These data demonstrate distinct, as yet unidentified pathways that transport different GPCRs to dendrites and spines, in which Rab43 plays a crucial role in the sorting and trafficking of the receptors likely via direct interaction. Support or Funding Information NIH R01 GM118915