Dopamine receptor-interacting protein 78 acts as a molecular chaperone for CCR5 chemokine receptor signaling complex organization.

Chemokine receptors are members of the G protein-coupled receptor (GPCR) family. CCR5 and CXCR4 act as co-receptors for human immunodeficiency virus (HIV) and several efforts have been made to develop ligands to inhibit HIV infection by blocking those receptors. Removal of chemokine receptors from the cell surface using polymorphisms or other means confers some levels of immunity against HIV infection. Up to now, very limited success has been obtained using ligand therapies so we explored potential avenues to regulate chemokine receptor expression at the plasma membrane. We identified a molecular chaperone, DRiP78, that interacts with both CXCR4 and CCR5, but not the heterodimer formed by these receptors. We further characterized the effects of DRiP78 on CCR5 function. We show that the molecular chaperone inhibits CCR5 localization to the plasma membrane. We identified the interaction region on the receptor, the F(x)6LL motif, and show that upon mutation of this motif the chaperone cannot interact with the receptor. We also show that DRiP78 is involved in the assembly of CCR5 chemokine signaling complex as a homodimer, as well as with the Gαi protein. Finally, modulation of DRiP78 levels will affect receptor functions, such as cell migration in cells that endogenously express CCR5. Our results demonstrate that modulation of the functions of a chaperone can affect signal transduction at the cell surface.

Dependence on different Rab GTPases for the trafficking of CXCR4 and CCR5 homo or heterodimers between the endoplasmic reticulum and plasma membrane in Jurkat cells.

Much is known about G protein coupled receptor trafficking and internalization following agonist stimulation. However, much less is known about outward trafficking of receptors from synthesis in the endoplasmic reticulum to the plasma membrane, or the role that trafficking might play in the assembly of receptor signaling complexes, important for targeting, specificity, and rapidity of subsequent signaling events. Up to now, very little is understood about receptor hetero-oligomers other than the fact that their assembly is done rapidly after biosynthesis. In our study we use bimolecular fluorescence complementation to selectively follow receptor dimers when expressed in Jurkat cells in order to clarify the trafficking itinerary those receptors follow to reach the plasma membrane and the resulting effect on signal transduction. CXCR4 and CCR5, previously shown to form both homo and hetero-oligomers, were used as our model to understand the specificities of trafficking along the anterograde pathway. The CXCR4 homodimer relies on Rabs2, 6 and 8 for anterograde transport regardless of the presence of endogenous CD4. The CCR5 homodimer relies on Rabs1 and 11 when CD4 is absent, but Rabs1 and 8 when CD4 was present. Interestingly, similar to the CCR5 homodimer, the CXCR4-CCR5 heterodimer relied on Rabs1 and 11 but also required Rab2 when CD4 was absent, and only Rab 1 when CD4 was present. Our results demonstrate that, although the receptors composing the heterodimeric complex are the same as in the homodimeric ones, the heterodimer traffics and signals differently than each homodimer. Our study demonstrates the importance of considering the receptor heterodimers as distinct signaling entities that should be carefully and individually characterized.