Date of Award


Embargo Period


Degree Type

Dissertation (CMU Access Only)

Degree Name

Doctor of Philosophy (PhD)


Biological Sciences


Manojkumar A. Puthenveedu


A defining feature of the eukaryotic cell is the abundance and diversity of membranous organelles. This allowed for a single cell to compartmentalize various functions and laid a foundation for the specialization of cell types in multicellular organisms. Greater complexity, however, requires more work to maintain, thus cells evolved machinery for directing the trafficking of proteins to their designated membranes. Early studies on membrane trafficking focused on understanding the biosynthetic pathway that takes proteins from their initial synthesis to be excreted or inserted in the plasma membrane. Transport of transmembrane proteins is not, however, a one way street. Many proteins, as a cycle of nutrient retrieval or signal activation, recycle from the surface through membrane compartments. Endosomes have risen as vital in the recycling and sorting process, but much of the mechanisms used remains unknown. Research into endosomal function has progressed in part by focusing on stereotypical receptors which can typify a class of cargo. Chief among these have been the non-recycling G-protein coupled receptor (GPCR) the δ opioid receptor (DOR), the bulk recycling transferrin receptor (TfR) and the sequence dependent recycling GPCR the β-2 adrenergic receptor (β2AR). The work in this document utilizes the β2AR to explore the intersection of membrane trafficking via early endosomes and intracellular signaling. We initially discovered that β2AR is selectively sorted to tubule microdomains associated with a dynamic actin cytoskeleton. This makes the recycling of active β2AR dependent on actin while bulk cargo can recycle independent of actin. We next connect β2AR actin dependence with the regulatory action of Protein Kinase A (PKA). Phosphorylation of β2AR via PKA in the cytoplasmic tail is necessary for restriction of β2AR to actin dependent tubules. Loss of phosohorylation by PKA led to defects in desensitization and recycling rate. Targeting actin to recycling tubules provides a powerful point of signaling regulation under control of signaling down-stream of β2AR itself. We wondered if this domain allowed for more than homeostatic control and shifted focus to endosomal actin as a potential regulator of sequence dependent recycling. We found the actin modifier cortactin, when phosphorylated by Src family kinases, promotes recycling of phosphorylated β2AR. Restriction of β2AR to and actin dependent pathway provides a link between β 2AR trafficking and the signaling cascades regulating actin. Having established the potential importance of interacting partners to β2AR trafficking we next began a search for the effector proteins which mediate the restriction of β2AR to actin dependent tubules. We concluded that such a binding partner would have to be localized to the body of the early endosome and selectively interact with the phosphorylated receptor. To capture such interacting proteins we optimized conditions for co-immunoprecipication and identified proteins enriched on internalized β2AR using stable isotope labeling of amino acids in cell culture (SILAC) with quantitative mass spectrometry. Bioinformatic analysis revealed proteins and complexes enriched in endosomes. While several known binding partners were detected we also detected a large number of proteins with little known function in mammalian cells. These novel interactions both suggest function for understudied proteins and allude to new regulatory mechanisms for endosomal sorting of β2AR.


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