Inefficient Endocytosis Limits Heterotrimeric G Protein Abundance on Endosomes

Kernekoncepter

Heterotrimeric G proteins are less abundant on endocytic vesicles compared to the plasma membrane, suggesting inefficient loading of G proteins onto internalized membranes.

Resumé

The study investigates the subcellular distribution of endogenous heterotrimeric G proteins in cultured HEK 293 cells using gene editing, confocal microscopy, and bioluminescence resonance energy transfer (BRET) techniques.
Key highlights:

G proteins are primarily localized on the plasma membrane and endolysosomes, with little to no detection on the endoplasmic reticulum, mitochondria, and Golgi apparatus.
G proteins are present on early, late, and recycling endosomes, but their density on endocytic vesicles is lower than on the plasma membrane, suggesting inefficient loading of G proteins onto internalized membranes.
Receptor activation does not significantly change the abundance of G proteins on endosomes, indicating that G protein endocytosis is not regulated by GPCR signaling.
The findings suggest that GPCR signaling from intracellular compartments may be disadvantaged by the lower density of G proteins on endosomes compared to the plasma membrane.

Statistik

Constitutive endocytosis supplies newly internalized endocytic vesicles with 20-30% of the G protein density found at the plasma membrane.
mNG-HRas ct density on FM4-64-positive endocytic vesicles was 64 ± 17% of the nearby plasma membrane.

Citater

"Constitutive endocytosis is sufficient to supply newly internalized endocytic vesicles with 20-30% of the G protein density found at the plasma membrane."
"Receptor activation does not change heterotrimer abundance on endosomes."

Vigtigste indsigter udtrukket fra

Visualization of endogenous G proteins on endosomes and other organelles

by Jang,W., Sen... kl. www.biorxiv.org 03-07-2024

https://www.biorxiv.org/content/10.1101/2024.03.05.583500v4

Dybere Forespørgsler

How might the uneven distribution of G proteins between the plasma membrane and endosomes impact the efficiency and dynamics of GPCR signaling from intracellular compartments?

The uneven distribution of heterotrimeric G proteins between the plasma membrane and endosomes can significantly impact the efficiency and dynamics of G protein-coupled receptor (GPCR) signaling from intracellular compartments. Since GPCR signaling is often initiated at the plasma membrane, where G proteins are most abundant, a lower density of G proteins on endosomes may limit the signaling capacity of internalized receptors. This is particularly relevant for receptors that continue to signal after endocytosis, as the reduced availability of G proteins on endosomes could lead to a slower or less effective signaling response.
Moreover, the study indicates that G proteins are present on early, late, and recycling endosomes but at a density that is only 20-30% of that found at the plasma membrane. This suggests that while some signaling can occur from endosomes, it may not be as robust as signaling from the plasma membrane. The dynamics of GPCR signaling could therefore be characterized by a rapid initial response at the plasma membrane followed by a diminished response as receptors are internalized, potentially leading to a temporal and spatial regulation of signaling pathways. This uneven distribution may also affect the downstream signaling cascades, as the availability of G proteins is crucial for the activation of various effectors, thereby influencing cellular responses to external stimuli.

What mechanisms could selectively enrich or exclude G proteins from nascent endocytic vesicles, and how might these mechanisms be regulated?

Several mechanisms could contribute to the selective enrichment or exclusion of G proteins from nascent endocytic vesicles. One proposed mechanism is steric occlusion, where the bulky structure of coat proteins and adapters involved in clathrin-mediated endocytosis may physically hinder the incorporation of G proteins into newly formed vesicles. This could be particularly relevant for heterotrimeric G proteins, which have larger extracellular domains compared to other membrane proteins.
Additionally, the study suggests that the dynamics of G protein endocytosis are not regulated by GPCR activation, indicating that the mechanisms governing G protein trafficking may be independent of receptor activity. However, it is possible that other regulatory proteins or pathways could modulate the loading of G proteins onto endocytic vesicles. For instance, the presence of specific lipid environments or membrane microdomains could influence the recruitment of G proteins to vesicles.
Furthermore, post-translational modifications, such as palmitoylation, could affect the membrane affinity of G proteins, thereby influencing their availability for incorporation into endocytic vesicles. The regulation of these mechanisms may involve signaling pathways that respond to cellular context, such as changes in membrane composition or the activation of specific kinases that modify G protein interactions with the membrane.

Given the important roles of G proteins in diverse cellular processes, how might the subcellular distribution of G proteins be altered in disease states or during cellular differentiation?

The subcellular distribution of G proteins can be significantly altered in disease states or during cellular differentiation, impacting their functional roles in signaling pathways. In cancer, for example, aberrant GPCR signaling is often associated with altered G protein expression and localization, which can lead to enhanced proliferation, survival, and metastasis. Changes in the expression levels of specific G protein subunits may result in a shift in the balance of signaling pathways, favoring those that promote tumorigenesis.
During cellular differentiation, the subcellular localization of G proteins may also change to support the specific signaling needs of differentiated cells. For instance, in neurons, the distribution of G proteins may be adapted to facilitate synaptic signaling and plasticity, with a potential increase in G protein presence at synaptic sites or within dendritic spines.
Moreover, in conditions such as heart disease or neurodegenerative disorders, the dysregulation of G protein signaling can lead to altered cellular responses to hormones and neurotransmitters, contributing to disease pathology. The study highlights that the low density of G proteins on endosomes may limit signaling from internalized receptors, suggesting that in disease states where GPCRs are overactive or misregulated, the altered distribution of G proteins could further exacerbate signaling imbalances, leading to pathological outcomes. Understanding these changes in G protein distribution could provide insights into therapeutic targets for modulating GPCR signaling in various diseases.