β-arrestin mediates communication between plasma membrane and intracellular GPCRs to regulate signaling

It has become increasingly apparent that G protein-coupled receptor (GPCR) localization is a master regulator of cell signaling. However, the molecular mechanisms involved in this process are not well understood. To date, observations of intracellular GPCR activation can be organized into two categories: a dependence on OCT3 cationic channel-permeable ligands or the necessity of endocytic trafficking. Using CXC chemokine receptor 4 (CXCR4) as a model, we identified a third mechanism of intracellular GPCR signaling. We show that independent of membrane permeable ligands and endocytosis, upon stimulation, plasma membrane and internal pools of CXCR4 are post-translationally modified and collectively regulate EGR1 transcription. We found that β-arrestin-1 (arrestin 2) is necessary to mediate communication between plasma membrane and internal pools of CXCR4. Notably, these observations may explain that while CXCR4 overexpression is highly correlated with cancer metastasis and mortality, plasma membrane localization is not. Together these data support a model where a small initial pool of plasma membrane-localized GPCRs are capable of activating internal receptor-dependent signaling events.


nature research | reporting summary
April 2020 Field-specific reporting Please select the one below that is the best fit for your research. If you are not sure, read the appropriate sections before making your selection. Reporting for specific materials, systems and methods Tissue samples of 2 species of palm and 9 associated beetle species visiting their flowers were collected across the geographical range of the species. Both insects and plants were sequenced by using double-digest RADseq. We used this data to delimit weevil species and then to fit models of isolation by environment, comparing results for the different insect species while considering the different kinds of interaction with host plants.
Samples were obtained from fieldwork in natural populations in 18 different localities across the known range of the palm species.
In each locality, insects visiting flowers were sampled by bagging a whole inflorescence and preserved in ethanol. In the lab, they were sorted to morphospecies and identified using the available literature. Plant tissues were obtained from leaf samples from the palm in which insects were sampled and other individuals in the population. In the lab we randomly selected up to 8 individuals per locality for DNA extraction and sequenced them by using double-digest RADseq. Additional individuals were sequenced after identification of cryptic species.
Collections were done between September and November (i. e. spring) of 2013 and 2014 across a few thousand kilometers throughout the range of both palm species.
Samples that resulted in very few sequencing reads were excluded from analysis. We also excluded insect species that were only observed in a few populations or for which there was little natural history data available by the time we did DNA sequencing.
A few samples were sequenced multiple times and in a previous manuscript we showed that they genotype calls were nearly identical (de Medeiros & Farrell 2018).
When more than 8 individuals for a species were available for sequencing for a given locality, we chose 10 individuals irrespective of their morphology. After finding out that the dataset included cryptic sympatric species, we sequenced new individuals choosing some that corresponded morphologically to the different putative species. We also randomized the position of samples in DNA extraction plates to avoid potential biases arising from cross-contamination when performing high-throughput automated DNA extractions.
After DNA extraction, samples were given numeric codes independent of their locality or species identity. Library preparation, sequencing and the initial steps in bioinformatics were done by referring to these codes only. In the case of cryptic species, we only scored morphological features after separating clusters based on the genetic data alone.
Fieldwork was done over a total of 4 months in 4 different trips. This was the onset of the rainy season across most of the region, and specific weather conditions varied.
Northeastern Brazil, in seasonally dry forest and coastal rainforest, between the states of Espírito Santo and Pernambuco.
Field sampling was done with SISBIO permit #39704-7 from Instituto Chico Mendes de Preservação da Biodiversidade, Brazil. Samples were deposited in collections in Brazil (Herbarium of the Institute of Biosciences, University of São Paulo and Museum of Zoology, University of São Paulo) and exported to the USA as loans.
The study involved bagging and shaking or excising inflorescences of Syagrus coronata and Syagrus botryophora, as well as pieces of folioles or whole leaves for samples deposited in herbaria. Both species have large geographical ranges and dense populations, producing several inflorescences per year. The disturbance, therefore, was minor. Climbing did not involve any piercing equipment that could damage plants.