Passage
Parathyroid hormone (PTH) is an important regulator of calcium and phosphate homeostasis. Specifically, PTH functions to increase plasma calcium concentration and reduce plasma phosphate levels. PTH secretion is closely regulated by changes in plasma calcium concentration, which are detected by the human calcium-sensing receptor (hCaSR) , a G protein-coupled receptor located on chief cells of the parathyroid gland. hCaSR activation ultimately inhibits PTH release by inducing a second messenger cascade in which intracellular calcium levels rise due to the hydrolysis of the membrane phospholipid phosphatidylinositol bisphosphate (PIP₂) into inositol trisphosphate (IP₃) and diacylglycerol (DAG) by the membrane-bound enzyme phospholipase C (PLC) .Secondary hyperparathyroidism is a condition characterized by excessive PTH release in response to depressed serum calcium or elevated serum phosphate levels. The novel peptide AMG 416, an hCaSR agonist, is being investigated as a possible treatment for secondary hyperparathyroidism caused by end-stage renal disease (ESRD) . IP-1, a downstream metabolite of IP₃, is a marker of hCaSR activity. Researchers used embryonic kidney cells lacking hCaSR (HEK-293T) and HEK-293T clone cells stably transfected with hCaSR (hCaSR) to measure IP-1 levels following AMG 416 treatment (Figure 1) .
Figure 1 IP-1 concentration in embryonic kidney cells lacking hCaSR (HEK-293T) and HEK-293T clone cells transfected with hCaSR (hCaSR) following AMG 416 treatmentThe dependence of AMG 416 on extracellular calcium was investigated by incubating hCaSR cells with increasing concentrations of AMG 416 in the presence or absence of calcium treatment (Figure 2) .
Figure 2 IP-1 levels in hCaSR cells incubated with and without calcium and AMG 416 peptide (Note: IP-1 levels are normalized to the number of cells present in each culture.) In addition, PTH concentration was measured in 5/6 Nx mice, an animal model of secondary hyperparathyroidism in human ESRD. Blood samples were taken from male and female 5/6 Nx mice for 8 hours following administration of varying concentrations of AMG 416 (Figure 3) , and PTH levels were measured.
Figure 3 Serum PTH concentration following administration of AMG 416 to 5/6 Nx mice
-Based on the passage and the experimental results shown in Figure 1, which event is an outcome of treating hCaSR cells with AMG 416?

Question

Passage
Parathyroid hormone (PTH) is an important regulator of calcium and phosphate homeostasis. Specifically, PTH functions to increase plasma calcium concentration and reduce plasma phosphate levels. PTH secretion is closely regulated by changes in plasma calcium concentration, which are detected by the human calcium-sensing receptor (hCaSR) , a G protein-coupled receptor located on chief cells of the parathyroid gland. hCaSR activation ultimately inhibits PTH release by inducing a second messenger cascade in which intracellular calcium levels rise due to the hydrolysis of the membrane phospholipid phosphatidylinositol bisphosphate (PIP₂) into inositol trisphosphate (IP₃) and diacylglycerol (DAG) by the membrane-bound enzyme phospholipase C (PLC) .Secondary hyperparathyroidism is a condition characterized by excessive PTH release in response to depressed serum calcium or elevated serum phosphate levels. The novel peptide AMG 416, an hCaSR agonist, is being investigated as a possible treatment for secondary hyperparathyroidism caused by end-stage renal disease (ESRD) . IP-1, a downstream metabolite of IP₃, is a marker of hCaSR activity. Researchers used embryonic kidney cells lacking hCaSR (HEK-293T) and HEK-293T clone cells stably transfected with hCaSR (hCaSR) to measure IP-1 levels following AMG 416 treatment (Figure 1) .

Figure 1 IP-1 concentration in embryonic kidney cells lacking hCaSR (HEK-293T) and HEK-293T clone cells transfected with hCaSR (hCaSR) following AMG 416 treatmentThe dependence of AMG 416 on extracellular calcium was investigated by incubating hCaSR cells with increasing concentrations of AMG 416 in the presence or absence of calcium treatment (Figure 2) .

Figure 2 IP-1 levels in hCaSR cells incubated with and without calcium and AMG 416 peptide (Note: IP-1 levels are normalized to the number of cells present in each culture.) In addition, PTH concentration was measured in 5/6 Nx mice, an animal model of secondary hyperparathyroidism in human ESRD. Blood samples were taken from male and female 5/6 Nx mice for 8 hours following administration of varying concentrations of AMG 416 (Figure 3) , and PTH levels were measured.

Figure 3 Serum PTH concentration following administration of AMG 416 to 5/6 Nx mice
-Based on the passage and the experimental results shown in Figure 1, which event is an outcome of treating hCaSR cells with AMG 416?

Quizplus · Accepted Answer

11ecba2d_0fbd_1fe8_a3bf_dbbd9addf3d4_MD0008_00 The passage states that the hCaSR is a G-protein coupled receptor (GPCR) whose activity is marked by IP-1, a downstream metabolite of IP₃. This indicates that hCaSR specifically activates the IP₃-DAG pathway. In addition, AMG 416 is an agonist of hCaSR, meaning it functions to bind and activate the receptor. As a result, the following processes would occur due to hCaSR activation by AMG 416: The G-protein heterotrimer (alpha, beta, and gamma subunits) attaches to the intracellular portion of the GPCR, a transmembrane cell surface receptor. When the ligand binds to the extracellular domain of the receptor, the GDP molecule initially bound to the G-protein alpha subunit is released, and a GTP molecule binds the G-protein in its place. The GTP-bound alpha subunit dissociates from the beta and gamma subunits, and activates the membrane-bound enzyme phospholipase C (PLC). PLC hydrolyzes the membrane phospholipid phosphatidylinositol bisphosphate (PIP₂) into inositol trisphosphate (IP₃) and diacylglycerol (DAG). Water-soluble IP₃ diffuses from the cell membrane through the cytoplasm to bind IP₃ receptors located on the endoplasmic reticulum and mitochondria. Binding of the IP₃ receptor opens calcium channels, allowing the release of stored calcium into the cytoplasm, which activates protein kinase C (PKC). DAG diffuses within the membrane and also activates PKC. PKC phosphorylates downstream intracellular proteins to produce its physiologic effects. (Choice A) The ligand of a GPCR binds to the extracellular region of the receptor. The G-protein is coupled to the receptor on its intracellular side, and therefore cannot be bound by the ligand, which in this case is calcium (not PTH). (Choice C) Calcium binds and activates PKC, not PLC. (Choice D) PLC is activated by the GTP-bound alpha subunit of the G-protein, not ATP. Educational objective: When the alpha subunit of a G-protein is bound to GDP, the protein is inactive. On ligand binding, GTP binds in place of GDP, causing activation of phospholipase C (PLC). Consequently, PLC breaks down phosphatidylinositol bisphosphate into diacylglycerol (DAG) and inositol trisphosphate (IP₃). IP₃ then triggers calcium release from the endoplasmic reticulum, and protein kinase C is subsequently activated by both DAG and calcium.

Passage Parathyroid Hormone (PTH) Is an Important Regulator of Calcium and and Phosphate