Download Case 26 The Role of Specific Amino Acids in the Peptide Hormone

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
C
C
Amino acid structure.
Signal transduction via G proteins.
Background
Glucagon is a 29-amino acid peptide hormone secreted by the pancreatic "-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 $-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 of action, 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 of the 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
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CASE 26 C The Role of Specific Amino Acids in Glucagon in Receptor Binding and Signal Transduction
studies have shown that an aspartate residue near the C-terminus of the receptor protein is essential for
glucagon binding.
Retaining the amino acid residues important for binding while modifying those amino acids involved
in signal transduction would result in a glucagon antagonist. Many such compounds have been
synthesized, but the search for the ideal antagonist has been complicated by the fact that several amino
acid residues in the glucagon molecule have been found to be important for both receptor binding and
signal transduction.
In the current study, the investigators used the technique of solid phase peptide synthesis to
synthesize modified glucagon molecules. They carried out two separate studies. The first study examined
the role of amino acid residues at positions 9, 15, and 21. The second study examined the role of amino
acid residues at positions 1,12, 17, and 18. In each study, amino acid residues were replaced with amino
acids with different properties, and the resulting analogs were tested for their ability to bind to liver
membrane receptors and carry out signal transduction. A true antagonist would be able to bind to
receptors while eliciting no response whatsoever. Analogs capable of binding with diminished (but not
abolished) activity are referred to as partial agonists.
Figure 26.1: Primary sequence of human glucagon.
1
16
1
His
Ser
2
Ser
Arg
3
4
5
6
Gln Gly Thr Phe
Arg Ala Gln Asp
7
8
Thr Ser
Phe Val
9
10
Asp Tyr
Gln Trp
11
Ser
Leu
12
Lys
Met
13 14
15
Tyr Leu Asp
Asn Thr
Questions
1.
Glucagon carries out its biological function by binding to extracellular hepatic receptors and then
putting into motion a series of events which leads to glycogenolysis. Draw a diagram which
describes the steps of this process.
2.
Why did the investigators choose amino acids at positions 1, 12, 17, and 18 for modification?
3.
The investigators synthesized a number of glucagon analogs which are listed in Table 26.2. The
ability of the glucagon analogs to bind to receptors and elicit a biological response was measured
and compared to native glucagon. Use the information provided in the table to answer the
following questions.
a. What is the effect of substituting or eliminating the amino acid at position 9?
b. What is the effect of the amino acid replacement or modification at position 12?
c. What is the effect of the amino acid replacement at position 17? Be specific.
d. What is the effect of the amino acid replacement at position 18? Be specific.
e. What is the role of the histidine at position 1?
4.
Write a summary paragraph describing the important findings of this study.
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CASE 26 C The Role of Specific Amino Acids in Glucagon in Receptor Binding and Signal Transduction
5.
Of the glucagon analogs presented here, which is the best glucagon antagonist? Could you design a
better glucagon antagonist than the analogs presented here? Explain the rationale for your design.
Table 26.1: Glucagon analogs with various amino acid replacements. Binding affinity refers to the
ability of the glucagon analog to bind to hepatic membrane receptors. Activity was measured by
testing each analog’s ability to stimulate cAMP production as compared to native glucagon. The
des prefix indicates that the specified amino acid has been deleted. (Based on Unson, et al., 1994
and 1998.)
Glucagon analog
Binding affinity
% of maximum activity
Glucagon
100%
100%
Des-Asp9
45%
8.3%
Lys9
54%
0%
N,-acetyl-Lys12
47
90.5
Ala12
17.3
59.7
Gly12
11.4
85.7
Glu12
1.0
80.4
Ala17
38
29
Leu17
30
88
Glu17
21.3
94.8
Ala18
13
94.4
Leu18
56
95
Glu18
6.2
100
Des-His1
63
44
Des-His1-Des-Asp9
7
0
Des-His1-Lys9
70
0
Des-His1-Glu12
0.11
28
Des-His1-Glu17
1.7
21.5
Des-His1-Glu18
0.44
18
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CASE 26 C The Role of Specific Amino Acids in Glucagon in Receptor Binding and Signal Transduction
References
Unson, C. G., Macdonald, D., Ray, K., Durrah, T. L., and Merrifield, R. B. (1991) J. Biol. Chem., 266,
pp. 2763-2766.
Unson, C. G., Wu, C.-R., and Merrifield, R. B. (1994) Biochemistry, 33, pp. 6884-6887.
Unson, C. G., Wu, C.-R., Cheung, C. P., and Merrifield, R. B. (1998) J. Biol. Chem. 273, pp. 1030810312.
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