the two connecting disulphide bonds can meet properly. Proinsulin is more soluble than
insulin, but has only about 35% insulin-like activity in vitro.
Insulin therefore consists of two peptide chains that are connected by two disulfide
bonds, since the C-peptide is cleaved off. There are some species-specific differences
in the amino acid sequence of the hormone. X-ray diffraction studies have shown that
insulin occurs as a hexameric protein containing two Zn atoms. The dimers are first
held by four hydrogen bonds and a hydrophobic bond along the β
21–30 sequence in the
form of an antiparallel β sheet. The dimers then bind by interaction of the B
14
–Ala,
B
7 –Leu, and B
18 –Val residues. The core of the hexamer contains water.
Structure–Activity Correlations. This detailed knowledge of the three-dimensional
structure of insulin led to the recognition that its biological activity resides in an area of
the molecule rather than in specific amino acid residues, just as dimerization and further association of the molecule also depend on an intact spatial structure. The foregoing concept is corroborated by structural modifications of the hormone. The last three
amino acids of the B chain can be removed without a loss of activity, but cleavage of
the C-terminal of the A chain (Asn
21 ) results in a total loss of activity. Amino acids can
be replaced inside the chains only if such substitution does not change the overall
geometry of the molecule. The structure–activity relationships of insulin derivatives are
inconsistent and not always comparable.
Characterization of Binding. Different equilibrium binding constants for insulin
have been reported by different authors. As measured with
125
I-labeled insulin in
adipocytes (fat cells), the K D varies from 5 × 10
−11 to 3 × 10
−9 M. There are about
10,000–12,000 binding sites in every fat cell. Although Scatchard plots indicate a single
population of receptors in fat cells, other cell populations (liver cells, lymphocytes)
show a Scatchard plot that is not linear. A possible explanation is based on the negative
cooperativity of the receptor, which implies that binding is inhibited and dissociation
accelerated in the presence of more insulin. This downregulation of insulin receptors
provides an intrinsic mechanism that enables certain cells to limit their response to
excessive or prolonged insulin concentrations.
The insulin receptor is composed of two heterodimers; each heterodimer is composed of an α unit and a β unit. The α unit is extracellular and contains the insulin
recognition and binding sites; the β unit spans the cellular membrane and contains a
tyrosine kinase. Although insulin can bind to a single αβ dimer, it binds with higher
affinity to the αβαβ tetrameric complex. When insulin binds to an α unit, the tyrosine
kinase associated with the corresponding β unit is stimulated. Following this, intracellular
proteins such as IRS-1 and IRS-2 (IRS=insulin receptor substrate) are phosphorylated
by the β subunit tyrosine kinase, and they in turn activate a network of phosphorylations
within the receptor cell.
Biochemical Effect. The biochemical effects of insulin include:
1. Facilitation of glucose transport into cells
2. Enhancement of intracellular glucokinase activity
3. Enhancement of amino acid incorporation into proteins
HORMONES AND THEIR RECEPTORS
365
insulin, but has only about 35% insulin-like activity in vitro.
Insulin therefore consists of two peptide chains that are connected by two disulfide
bonds, since the C-peptide is cleaved off. There are some species-specific differences
in the amino acid sequence of the hormone. X-ray diffraction studies have shown that
insulin occurs as a hexameric protein containing two Zn atoms. The dimers are first
held by four hydrogen bonds and a hydrophobic bond along the β
21–30 sequence in the
form of an antiparallel β sheet. The dimers then bind by interaction of the B
14
–Ala,
B
7 –Leu, and B
18 –Val residues. The core of the hexamer contains water.
Structure–Activity Correlations. This detailed knowledge of the three-dimensional
structure of insulin led to the recognition that its biological activity resides in an area of
the molecule rather than in specific amino acid residues, just as dimerization and further association of the molecule also depend on an intact spatial structure. The foregoing concept is corroborated by structural modifications of the hormone. The last three
amino acids of the B chain can be removed without a loss of activity, but cleavage of
the C-terminal of the A chain (Asn
21 ) results in a total loss of activity. Amino acids can
be replaced inside the chains only if such substitution does not change the overall
geometry of the molecule. The structure–activity relationships of insulin derivatives are
inconsistent and not always comparable.
Characterization of Binding. Different equilibrium binding constants for insulin
have been reported by different authors. As measured with
125
I-labeled insulin in
adipocytes (fat cells), the K D varies from 5 × 10
−11 to 3 × 10
−9 M. There are about
10,000–12,000 binding sites in every fat cell. Although Scatchard plots indicate a single
population of receptors in fat cells, other cell populations (liver cells, lymphocytes)
show a Scatchard plot that is not linear. A possible explanation is based on the negative
cooperativity of the receptor, which implies that binding is inhibited and dissociation
accelerated in the presence of more insulin. This downregulation of insulin receptors
provides an intrinsic mechanism that enables certain cells to limit their response to
excessive or prolonged insulin concentrations.
The insulin receptor is composed of two heterodimers; each heterodimer is composed of an α unit and a β unit. The α unit is extracellular and contains the insulin
recognition and binding sites; the β unit spans the cellular membrane and contains a
tyrosine kinase. Although insulin can bind to a single αβ dimer, it binds with higher
affinity to the αβαβ tetrameric complex. When insulin binds to an α unit, the tyrosine
kinase associated with the corresponding β unit is stimulated. Following this, intracellular
proteins such as IRS-1 and IRS-2 (IRS=insulin receptor substrate) are phosphorylated
by the β subunit tyrosine kinase, and they in turn activate a network of phosphorylations
within the receptor cell.
Biochemical Effect. The biochemical effects of insulin include:
1. Facilitation of glucose transport into cells
2. Enhancement of intracellular glucokinase activity
3. Enhancement of amino acid incorporation into proteins
HORMONES AND THEIR RECEPTORS
365