The recruitment of certain GPCRs preferentially to the ciliary membrane could be mediated by:
- Dedicated pathways driving localization of these receptors (such as the Tulp3-IFT-A complex.
- Hijacking of canonical trafficking mechanisms from the golgi to the cellular membrane.
- Retention factors in the ciliary compartment. A general strategy outlining the workflow for studying GPCR trafficking mechanisms is described in the research overview section.
To begin with, we would utilize TAP-MS approaches with membrane-tagged ciliary localization signals (CLS) of Gpr161 in order to discover associated cellular factors, not only important in trafficking but also in downstream signaling. An initial interaction network of the membrane domain-tagged CLS of Gpr161, in addition to networks of associated proteins has already been generated. We would further characterize the importance of these factors in ciliary localization of Gpr161 by doing RNAi-based knockdown studies on endogenous Gpr161 levels in cultured cells. The core facilities for high-content screening at UTSW would also provide us with excellent opportunities to implement supplementary whole-genome RNAi-based approaches for characterizing factors important in localization and downregulation of ciliary GPCRs. The proteomic approaches would also be extended to additional CLS from other ciliary GPCRs, in order to define the common logic behind these apparently disparate domains mediating ciliary trafficking.
Deciphering the mechanisms for down-regulation of ciliary receptors is equally important in understanding their regulation in cellular contexts. Our discovery of the regulation of ciliary levels of Gpr161 by Shh signaling provides the unique opportunity to discover pathways important in these processes. Defining regions in this GPCR, and the cellular mechanisms underlying its Shh-mediated internalization would be an important focus of our lab in the near future.
For more information about Dr. Saikat Mukhopadhyay's research, please visit his faculty profile.