Case 26 The Role of Specific Amino Acids in the Peptide Horm

Case 26 The Role of Specific Amino Acids in the Peptide Hormone Glucagon in Receptor Binding and Signal Transduction Focus concept Amino acid side chains important in glucagon binding and signal transduction are identified. Prerequisites Amino acid structure. Signal transduction via G proteins. Background Glucagon is a 29-amino acid peptide hormone secreted by the pancreatic a-cells in response to low glucose concentrations. Its primary amino acid sequence is shown in Table 26.1. Glucagon acts primarily on the liver where binding to specific extracellular receptors stimulates glycogenolysis and gluconeogenesis with subsequent release of glucose from the liver for the benefit of other body tissues Glucagon is counter-regulatory to insulin which is secreted by pancreatic A-cells and stimulates cellular uptake of exogenous glucose from the blood. During feeding insulin levels are high and glucagon levels are low. The opposite is true during fasting glucagon levels rise and insulin concentrations decrease The glucagon hormone has been the subject of much research interest in decades past, not just because of its importance in carbohydrate metabolism, but also because its mechanism ofaction, via the activation of a G-protein-linked enzyme, is a model for signal transduction. But more recently attention has focused on the role of glucagon in the disease diabetes mellitus. Several studies have shown that in diabetics the lack of insulin is accompanied by hypersecretion of glucagon. The excess glucagon secretion leads to release of glucose from the liver, which exacerbates the high blood glucose concentrations in the untreated diabetic. Diabetics are currently treated with exogenous insulin. But some investigators have suggested that the treatment regimen ofthe diabetic should address the glucagon hypersecretion as well as the lack of insulin. One way to do this would be to administer a glucagon antagonist along with insulin. A glucagon antagonist is a molecule that would be able to bind to extracellular receptors on liver cells, but would be unable to carry out the signal transduction process. The glucagon antagonist would compete for binding with endogenous glucagon. If the antagonist bound instead of the endogenous glucagon, glycogenolysis would not occur. In order to construct a glucagon antagonist it is necessary to determine exactly which amino acids contribute to receptor binding and which amino acids are involved in signal transduction. These experiments were first carried out in the mid-seventies, but recent advances in biotechnology have facilitated the process. For example, the glucagon receptor gene has been cloned and sequenced, and

Solution

What is the effect of substituting or eliminating the amino acid at position 9?

Here, when Asp was removed from position 9, it has resulted in binding affinity of 45% and % of maximum activity is 8.3%, but when both His1 and Asp9 were removed together it resulted in the binding affinity of 7 with % of maximum activity of 0.

When Asp was replace with Lys at position 9, then the binding affinity remained at 70, but % of maximum activity was 0

What is the effect of the amino acid replacement or modification at position 12?

At 12th position in glucagon Lys is located, when it was replaced with Ala, there is a binding affinity of 17.3 with % of max activity of 59.7%, and with Gly substitution it to binding affinity of 11.4 and % of maximum activity of 85.7 and Glu it got binding affinity of 1 and % maximum activity of 80.4%.

What is the effect of the amino acid replacement at position 17? Be specific.

At 17th position we are having arginine, when we replaced this with Ala, it has resulted in the binding affinity of 38 and % of max affinity of 29, with Leu replacement got a binding affinity of 30 and % of max affinity binding of 88, with Glu replacement got a binding affinity of 21.3 and binding affinity of 94.8.

What is the effect of the amino acid replacement at position 18? Be specific.

At position 18 also we are having arginine, with Ala substitution here got a binding affinity of 13 and % of max binding affinity of 94.4 and with Lue substitution got a binding affinity of 56 and % max affinity of 95, with Glu substitution got a binding affinity of 6.2 with % of maximum affinity of 100.

What is the role of the histidine at position 1?

When we deleted Histidine, it has resulted in the binding affinity of 63 and % of max affinity of 44, but deletion in combination with Asp9 deletion resulted in the binding affinity of 7 with % of max activity of 0. This clearly shows His1 plays an important role in the binding process to the receptor.

Write a summary paragraph describing the important findings of this study.

Through this study, the importance of aspartic acid located at position 9 has come to know, that this amino acid is critical for transduction and it dictates either receptor recognition or biological activity.

The amino acid Asp9 will try to operate in conjunction with His1 for the binding and activation of the glucagon receptor. If we replace these aspartic acid or Histidine with some other amino acid, it has affected the binding and transduction process.

When we deleted His1 and Asp9, it has resulted in 0 % of max activity, this clearly shows that analogue with this deletion can stop the transduction process and deletion of His1 has clearly showed a decrease in the binding affinity and in combination in a loss of % max activity