The F6LL motif in the Cterminus, the RRR motif in the third intracellular loop, and an isolated Leu residue in the ICL1, which are essential for export trafficking of a2B-AR. In a continuing effort to search for the structural determinants of a2-AR transport, we expanded our studies to define the role of the ICL1 in the cell-surface expression of a2A-AR. Surprisingly we found that, in addition to Leu residue, a neighboring Lys residue specifically modulates the ER export and cell-surface expression of a2A-AR and this function is likely dictated by its positively charged property. These data provide the first evidence indicating that the ICL1 may possess multiple signals that use distinct mechanisms to control the processing of a2AAR. The molecular mechanisms underlying export from the ER and subsequent transport to the cell surface of the GPCR superfamily remain poorly elucidated. It has been significant efforts to define the structural determinants for GPCR export and several highly conserved hydrophobic and charged sequences which are required for GPCR export from the ER to the cell surface have been identified. We have previously investigated the role of the ICL1 in the cell-surface transport of GPCRs and identified a Leu residue essential for ER export of a group of GPCRs, including a2B-AR. This Leu residue is extremely conserved amongst the family A GPCRs and indeed, as Adriamycin demonstrated in this manuscript, Leu64 in the ICL1 also plays an obligatory role in a2A-AR export from the ER and transport to the cell surface. Thus, this isolated Leu may provide a common signal directing anterograde transport of multiple nascent GPCRs. The most important finding described in this manuscript is that different positively charged residues on the ICL1 have differential impacts on the cell-surface transport of distinct GPCRs. During the studies on the function of the ICL1. First, intact cell ligand binding and flow cytometry to quantify the cell-surface receptors showed that mutation of Lys65 to Ala reduced a2A-AR cell-surface number by more than 50%. Second, the significant reduction of receptor expression at the cell surface was supported by direct visualization of intracellular localization of the mutated receptors. As revealed by confocal microscopy, GFP-tagged K65A was extensively expressed inside the cell which is in contrast to robust cell-surface expression of its wild-type counterpart. Third, the function of Lys65 in controlling a2A-AR transport is also supported by receptor-mediated signal propagation as measured by ERK1/2 activation. The reduction of ERK1/2 activation in cells expressing K65A as compared with cells expressing wild-type a2A-AR could be simply due to less expression of the mutated a2AAR at the cell surface, which are available for binding to the ligand, indicating that mutation of Lys65 not only reduced the cellsurface expression of a2A-AR but also attenuated receptormediated signal propagation. These data strongly indicate that, in addition to Leu64, Lys65 in the ICL1 also plays a crucial role in regulating cell-surface transport of a2A-AR. By contrast, mutation of the positively charged residue at the same position, Arg49, did not influence the cell-surface transport of a2B-AR. These data demonstrated for the first time that a single positively charged residue on the short ICL1 is involved in the regulation of export trafficking of GPCRs in a receptor subtype-specific fashion. It is likely that Lys65 residue modulates a2A-AR transport at the level of the ER. We found that the a2A-AR mutant K65A was extensively co-localized with the ER marker DsRed2-ER, suggesting that the mutant was unable to exit from the ER where they are synthesized. Therefore, these studies provide another novel regulatory mechanism for the ER export of nascent a2A-AR.