Essential Endocrinology and Diabetes. Richard I. G. Holt

Figure 3.2 Basic components of a membrane‐spanning cell‐surface receptor. The hormone acts as ligand. The ligand‐binding pocket in the extracellular domain tends to be comparatively rich in cysteine residues that form internal disulphide bonds as part of a precise three‐dimensional folded structure. For some hormones, e.g. growth hormone, the extracellular domain can be cleaved and circulate as a potential binding protein. Circulating fragments of the thyroid‐stimulating hormone receptor can be immunogenic leading to auto‐antibody formation in autoimmune thyroid disease. The α‐helical membrane‐spanning domain is rich in hydrophobic and uncharged amino acids. The C‐terminal cytoplasmic domain either contains, or links to, catalytic sites, which initiate intracellular signalling stimulated by hormone binding.

Box 3.2 Binding characteristics of hormone receptors

       High affinity: hormones circulate at relatively low concentrations – receptors are like ‘capture systems’

       Reversible binding: one reason for the transient nature of endocrine responses

       Specificity: receptors distinguish between closely related molecular structures

Protein phosphorylation is a key molecular switch. Approximately 10% of proteins are phosphorylated at any given time in a mammalian cell. The phosphate group is donated from ATP during catalysis by the kinase enzyme. It is accepted by the polar hydroxyl group of the amino acids, serine, threonine or tyrosine (Figure 3.5a) and causes a conformational change in the three‐dimensional shape of the protein (Figure 3.5b). In many signalling cascades, the phosphorylated protein can also act as a kinase and phosphorylate the next protein in the sequence. This relays and amplifies the intracellular signal generated by the hormone binding to its receptor (Figure 3.5c).

Figure 3.3 Hormone–receptor systems are saturable. Increasing amounts of labelled hormone are incubated with a constant amount of receptor. The amount of bound labelled hormone increases as more is added until the system is saturated. At this point, further addition of hormone fails to increase the amount bound to receptors. The concentration of hormone that is required for half‐maximal saturation of the receptors is equal to the dissociation constant (K _D) of the hormone–receptor interaction.

Figure 3.4 Hormone–receptor interactions are reversible. Constant amounts of labelled hormone and receptors are incubated together for different times. The bound label increases with time until it reaches a plateau, when the bound and free hormone has reached a dynamic equilibrium. In a dynamic equilibrium, hormone continually associates and dissociates from its receptor. Adding excess unlabelled hormone competes for access to the receptors. Consequently, the amount of bound labelled hormone decreases with extended incubation (dashed line).

Box 3.3 Categories of cell‐surface receptors

       Tyrosine kinase receptors

       Signal via phosphorylation of the amino acid, tyrosine

       G‐protein–coupled receptors

       Activate or inhibit adenylate cyclase and/or phospholipase C (PLC)

       Signal via second messengers: cyclic adenosine monophosphate (cAMP), inositol triphosphate (IP3), diacylglycerol (DAG) and intracellular calcium

       Signal via phosphorylation of serine and threonine amino acids

Tyrosine kinase receptors

      Phosphorylation of tyrosine kinase receptors can occur through:

       tyrosine kinase that is intrinsic to the cytosolic domain of the receptor and activated after hormone–receptor binding; or

       separate tyrosine kinases that are recruited to the intracellular portion of the receptor after hormone binding (Figure 3.1).

      By either mechanism, the conformational change induced by phosphorylation creates ‘docking’ sites for other proteins. Frequently, this occurs via conserved motifs within the target protein, known as ‘SH2’ or ‘SH3’ domains. These domains may be involved in the activation of downstream kinases or they may stabilize other signalling proteins within a phosphorylation cascade.

      Receptors with intrinsic tyrosine kinase activity

      Intrinsic tyrosine kinase receptors auto‐phosphorylate upon binding of the appropriate hormone. This group includes the receptors for insulin, epidermal growth factor (EGF), fibroblast growth factor (FGF) and insulin‐like growth factor I ( IGF‐I). EGF and FGF receptors exist as monomers that dimerize upon hormone binding. The dimerization activates tyrosine phosphorylation. Those for insulin and IGF‐I exist in their unoccupied state as preformed dimers. The signalling pathways for all these receptors are heavily involved in cell growth and proliferation.

       Insulin signalling pathways

The dimerized insulin receptor comprises two α‐ and two β‐subunits linked by a series of disulphide bridges (Figure 3.6; Chapter 11). When insulin binds, auto‐phosphorylation occurs on the cytosolic domains of the β‐subunit. The activated receptor then phosphorylates two key intermediaries, insulin receptor substrate (IRS) ‐1 or ‐2, which are thought to be essential for almost all the biological actions of insulin. IRS1 has many potential tyrosine phosphorylation sites, at least eight of which are phosphorylated by the activated insulin receptor.

Multiple phosphorylation of IRS‐1 or ‐2 leads to the docking of several proteins with SH2 domains, and the activation of divergent intracellular signalling. For example, docking of phosphatidylinositol‐3‐kinase (PI3‐kinase) leads to deployment of the glucose transporter (GLUT) family members. For instance, in adipose tissue and muscle, GLUT‐4 translocates from intracellular vesicles to the cell membrane, to facilitate glucose uptake into the cell. The mitogenic