The PDZ Ligand of Somatostatin Receptor 2 (sst2) Acts As an Intracellular Trafficking Signal
Presentation Number: SUN 246
Date of Presentation: April 2nd, 2017
Courtney Olsen*1 and Agnes Schonbrunn2
1University of Texas McGovern Medical School, Houston, TX, 2Univ of Texas-McGovern Med School, Houston, TX
Sst2 is a Gi-coupled G-protein coupled receptor (GPCR) that is highly expressed on the cell surface of many neuroendocrine tumors and is the target of Octreotide and Lanreotide, the primary medical treatments for acromegaly. Sst2 is internalized into clathrin-coated pits, traffics through early endosomes, and is rapidly recycled to the plasma membrane following agonist removal. However, little is known about the pathways involved in sst2 recycling. PDZ ligands, through interaction with PDZ proteins, regulate the intracellular trafficking of numerous GPCRs. Sst2 contains a C-terminal PDZ ligand which can bind to several PDZ proteins but its role in receptor trafficking and regulation is not known. We hypothesized that the sst2 PDZ ligand acts as a receptor sorting and recycling signal. To test this hypothesis, we created sst2 receptor mutants lacking either 3 or 10 C-terminal amino acids. We found that these mutants are fully functional: they express at the plasma membrane, respond to agonist, and couple to Gi, all similarly to wild type sst2. To test the sst2 PDZ ligand’s effect on trafficking, we used cell surface receptor ELISAs to measure the rate and extent of agonist induced internalization and recycling. For the recycling assays, cells were treated with agonist to induce maximal receptor internalization followed by agonist removal to allow receptor recovery for various times up to 1hr. Surprisingly, both receptor mutants internalized and rapidly recycled to the plasma membrane to the same extent as wild type. Next we created a chimeric receptor to determine if the sst2-PDZ ligand is capable of acting as an intracellular trafficking signal. The Delta Opioid Receptor (DOR) is also a Gi-coupled GPCR and has been shown to undergo agonist-induced endocytosis via the same pathway as sst2 and we confirmed this by immunocytochemistry. However, after sorting at the endosome, DOR is trafficked to lysosomes to be degraded rather than recycled to the plasma membrane. We created a chimeric receptor (DOR-sst2), adding the last 10 amino acids of sst2 to DOR to determine if these residues were sufficient to alter DOR from a non-recycling to a recycling receptor. Using the ELISA described above, we found that DOR and DOR-sst2 internalized to the same extent after treatment with DADLE. However, 1h after agonist removal DOR-sst2 recycled to 73±20% of non-treated cells compared to wild type DOR, which recovered only to 31±12%. Our results indicate the sst2 PDZ ligand can act as an intracellular trafficking signal and we hypothesize that sst2 can recycle to the plasma membrane through multiple pathways depending on PDZ domain interactions. The trafficking and regulation of sst2 is thought to be of critical importance for tumor responsiveness to somatostatin agonists and therefore sst2 PDZ ligand-domain interactions are likely to play an important role in the treatment of acromegaly.
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