Abstract P238: Regulation Of G Protein-coupled Receptor Kinase 4 Expression By Serum In Breast Cancer And Renal Proximal Tubule Cells

G protein-coupled receptor kinase 4 (GRK4) is a member of the GRK family which play critical role in regulation of the function of G protein-coupled receptors. Our previous studies have shown that GRK4 not only plays a role in regulating sodium excretion in renal proximal tubule cells but also acts as a stimulator on proliferation of breast cancer cells. Uncontrolled proliferation is a characteristics of cancer cells and GRK4 is upregulated in breast cancer cells. We hypothesized that expression of GRK4 may be regulated differently in cancer and non-cancer cells. To test this hypothesis, expression of GRK4 in response to serum was compared in breast cancer cells and renal proximal tubule cells by Western analysis. In three breast cancer cell lines serum withdrawal caused rapid reduction in the levels of GRK4 which occurred as early as 15 min. GRK4 levels correlated with the concentrations of serum added to the culture media. To determine if growth factors were a critical element for maintaining GRK4 levels in the cells, EGF (10-20 ng/ml) was added to serum free medium for 24 h. There was no increase in GRK4 levels in the cells treated with EGF compared with the serum starvation control. Similarly, serum withdrawal (16 h) led to 40-80% decrease of GRK4 levels in renal proximal tubule cells even in the presence of EFG supplement. Serum feeding for 30 min after starvation dramatically increased the levels of GRK4 in both breast cancer cells and RPTC which exceeded the steady state levels. This rapid recovery of GRK4 protein do not need de novo protein synthesis because pretreatment of the cells with protein synthesis inhibitor, cycloheximide (10 μg/ml, 24 h), did not prevent this event. Expression of GRK2, another member of the GRK family, was not affected by serum starvation. Our results have shown that GRK4 is very sensitive to serum concentration in breast cancer cells as well as in RPTC. Preliminary studies suggest that rapid protein degradation rather than shutting down the protein synthesis plays a major role in this kind of GRK4 regulation. The biological significance of serum regulation of GRK4 in cancer and non-cancerous cells needs further investigation.

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G protein‐coupled receptor 37L1 participates in epigenetic gene regulation in human renal proximal tubule cells

The FASEB Journal ◽

10.1096/fasebj.2020.34.s1.06274 ◽

2020 ◽

Vol 34 (S1) ◽

pp. 1-1

Author(s):

Prasad R. Konkalmatt ◽

Caini Fan ◽

Santiago Cuevas ◽

Ines Armando ◽

Pedro A. Jose

Keyword(s):

Gene Regulation ◽

Proximal Tubule ◽

G Protein ◽

Renal Proximal Tubule ◽

G Protein Coupled Receptor ◽

Proximal Tubule Cells ◽

Tubule Cells ◽

Epigenetic Gene Regulation ◽

Renal Proximal Tubule Cells ◽

G Protein Coupled

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Abstract 653: G-protein-coupled Receptor Kinase 4 Binds Preferentially To The Caveolin-1α Isoform In Rat Renal Proximal Tubule Cells

Hypertension ◽

10.1161/hyp.60.suppl_1.a653 ◽

2012 ◽

Vol 60 (suppl_1) ◽

Author(s):

Dora Bigler Wang ◽

John J Gildea ◽

Robin A Felder

Keyword(s):

Proximal Tubule ◽

G Protein ◽

Renal Proximal Tubule ◽

Receptor Kinase ◽

G Protein Coupled Receptor ◽

Proximal Tubule Cells ◽

Tubule Cells ◽

Renal Proximal Tubule Cells ◽

G Protein Coupled ◽

Caveolin Scaffolding Domain

Caveolins, localized in lipid rafts of plasma membranes, tether and regulate signaling complexes into functional units and have been shown to inhibit g-protein-coupled receptor kinases GRK2 and GRK5 via the caveolin scaffolding domain. In human renal proximal tubule cells (RPTC), the dopamine-1-receptor (D1R) is phosphorylated and inhibited by G protein-coupled receptor kinase 4 (GRK4). Defects in D1R coupling leads to sodium-retention and a rise in blood pressure. We showed earlier that chronic disruption of lipid rafts or Caveolin-1 (Cav1) protein expression in the kidney induces hypertension in rats by relieving the steric inhibition of Cav1 on GRK4. Here we extend these studies by examining caveolin expression in renal proximal tubule cells microdissected from Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR). We discovered a significant decrease in Cav1 and Cav2 expression in SHR compared to WKY (98 ±1.2% reduction; n=3, P<0.001). In order to determine if the D1R coupling defect found in these SHR renal proximal tubule cells is due to loss of either of these proteins, we made stable SHR cell lines expressing either Cav1α, Cav1β or Cav2. Using co-immunoprecipitiation and western blot analysis, we demonstrate for the first time that GRK4 and Cav1 physically interact in rat cells, and that it is the Cav1α isoform that binds predominantly to GRK4 (Cav1β, Cav2 and vector control show a 90 ±1.5%, 84±0.8% and 87±2.1% reduction, respectively; n=3, P<0.001). Similar to an effect found in mouse Cav1 knockout animals, we find that Cav2 is not stable without the co-expression of Cav1. Our data suggest that Cav1α. but not Cav1β, is important for normal D1R function by binding to and inhibiting GRK4, implicating a domain other than the caveolin scaffolding domain in this interaction. These findings might be used in designing selective GRK4 inhibitors as novel antihypertensive compounds.

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G protein-coupled receptor 37L1 regulates renal sodium transport and blood pressure

AJP Renal Physiology ◽

10.1152/ajprenal.00289.2018 ◽

2019 ◽

Vol 316 (3) ◽

pp. F506-F516 ◽

Cited By ~ 3

Author(s):

Xiaoxu Zheng ◽

Laureano D. Asico ◽

Xiaobo Ma ◽

Prasad R. Konkalmatt

Keyword(s):

Proximal Tubule ◽

G Protein ◽

Renal Proximal Tubule ◽

Intracellular Sodium ◽

Proximal Tubule Cells ◽

Tubule Cells ◽

Renal Proximal Tubule Cells ◽

And Function ◽

G Protein Coupled ◽

The Brain

G protein-coupled receptors (GPCRs) in the kidney regulate the reabsorption of essential nutrients, ions, and water from the glomerular filtrate. Abnormalities in renal epithelial ion transport play important roles in the pathogenesis of essential hypertension. The orphan G protein-coupled receptor 37L1 (GPR37L1), also known as endothelin receptor type B-like protein (ETBR-LP2), is expressed in several regions in the brain, but its expression profile and function in peripheral tissues are poorly understood. We found that GPR37L1 mRNA expression is highest in the brain, followed by the stomach, heart, testis, and ovary, with moderate expression in the kidney, pancreas, skeletal muscle, liver, lung, and spleen. Immunofluorescence analyses revealed the expression of GPR37L1 in specific regions within some organs. In the kidney, GPR37L1 is expressed in the apical membrane of renal proximal tubule cells. In human renal proximal tubule cells, the transient expression of GPR37LI increased intracellular sodium, whereas the silencing of GPR37LI decreased intracellular sodium. Inhibition of Na+/H+ exchanger isoform 3 (NHE3) activity abrogated the GPR37L1-mediated increase in intracellular sodium. Renal-selective silencing of Gpr37l1 in mice increased urine output and sodium excretion and decreased systolic and diastolic blood pressures. The renal-selective silencing of GPR37L1 decreased the protein expression of NHE3 but not the expression of Na+-K+-ATPase or sodium-glucose cotransporter 2. Our findings show that in the kidney, GPR37L1 participates in renal proximal tubule luminal sodium transport and regulation of blood pressure by increasing the renal expression and function of NHE3 by decreasing cAMP production. The role of GPR37L1, expressed in specific cell types in organs other than the kidney, remains to be determined.

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