Melatonin Binding Sites
In the late 1970’s binding of tritiated
melatonin (3H-melatonin) in the bovine and chicken brain was
reported (Cardinali et al. 1979; Cohen et al. 1978; Niles et al. 1979).
However due to its low specific activity it was replaced by 2[125I]-iodomelatonin,
which showed higher specific activity (Vakkuri et al. 1984). While 2[125I]-iodomelatonin is
still used, a more detailed analysis has demonstrated that [3H]-melatonin
better resembles the normal binding and dissociation kinetics of melatonin then
does 2[125I]-iodomelatonin (Kennaway et al. 1994). Based
on the results using 2[125I]-iodomelatonin, the existence of two
distinct melatonin receptor sites was shown (Pang et al. 1993; Pickering and
Niles 1989; Dubocovich 1995; Sugden et al. 1997). The first was a high affinity site (sub-divided into MT1 and MT2)
and the second was a low affinity site (described as MT3).
Some
non-receptor binding sites for melatonin were found on both GABAA
receptors (Coloma and Niles 1988) and K+ channels (Varga et al.
2001), and they will be discussed later.
The responses to melatonin can be categorized into two groups: receptor-
and non-receptor mediated (discussed below).
The classification of melatonin responses can also be differentiated
according to the concentration of melatonin used to elicit them. While the effects of low (picomolar)
concentrations of melatonin have been considered physiological, the effects
observed following application of micro- to millimolar concentrations is
usually recognized as pharmacological.
While this arbitrary classification is still predominantly used
(Tricoire et al. 2002) one has to be aware that high concentrations possibly
even in the micromolar may occur normally in vivo and still be
considered physiological (Reiter 2002).
For an action of melatonin to be
categorized as occurring at the melatonin receptor, it must meet the following
criteria: 1) the binding of melatonin must be with high affinity and
selectivity; 2) melatonin binding should be saturable and reversible, reaching a
time-dependent equilibrium; and 3) binding of melatonin must elicit a
biological response. These criteria can
be tested experimentally with the use of two specific methods, radioligand
binding and cAMP functional assay.
Specific
binding using 2[125I]-iodomelatonin has been demonstrated to be at
three melatonin receptors: MT1 (formerly Mel1a; Dubocovich et al. 1998), MT2
(formerly Mel1b; Dubocovich et al. 1998) and MT3 (formerly Mel1c and more
recently described as quinone reductase 2; Nosjean et al. 2000). Radioligand binding is still one of the most
effective methods to demonstrate the presence of melatonin receptors (Duncan et
al. 1986; Dubocovich and Takahashi 1987; Siuciak et al. 1991).
Activation of
MT1 and MT2 receptors, by low picomolar concentrations of melatonin (1-10 pM)
leads to a decrease in adenylyl cyclase activity via a Gi receptor (Figure
1.3; Reppert 1997; Reppert et al. 1994; Godson and Reppert 1997; Carlson et
al. 1989; Shiu et al. 1989; Conway et al. 1997), while the activation of MT3
initiates hydrolysis of phosphatidylinositide (Eison and Mullins 1993; Blumenau
et al. 2001; Mullins and Eison 1994). MT1
receptor mRNA has been found in brain structures including the SCN, pars
tuberalis, hypothalamus, cerebellum, hippocampus and cerebral cortex of mammals
(Mazzucchelli et al. 1996;
Figure 1.3. The function of MT1 and MT2 melatonin receptors.
Both MT1 and MT2 melatonin receptors are shown in
the plasma membrane coupled to a Gi-protein. Activation of these receptors by the ligand
leads to a decrease in the production of cAMP by adenylate cyclase (AC). alpha (a), beta (b),
and gamma (g) subunits are the functional components of the
G-protein. 
- inhibition.
Bittman and Weaver
1990).
2-Imel is the best agonist for this receptor followed by melatonin and
6Cl-melatonin. The MT2 receptor has
been localized in the mammalian retina and hippocampus (Mushoff et al. 2002;
Dubocovich et al. 1997). The most
potent agonist for the MT2 is melatonin followed by 6-ClMel and 2-Imel. The MT3 receptor was originally found in the
brain, testes, and kidneys of gerbils (Paul et al. 1999). 2-Imel and 6-ClMel have a higher affinity
for the receptor then melatonin itself (Zawilska and Novak 1999). In opposite to MT1 and MT2 receptors, which
demonstrate a high affinity binding to melatonin (Kd = 10-200 pM), the ability
of MT3 to bind melatonin is much lower (Kd = 3-9 nM). The MT3 has recently been identified as quinone reductase 2
(Nosjean et al. 2000), which functions as an oxidoreductive enzyme. It is currently unclear how MT3 sites, which
lead to phosphatidylinositol turnover, can simultaneously function as quinone
reductase 2.
Functional
cAMP assay
Binding of
melatonin fulfills all criteria for binding to a receptor site. It is time- and
temperature -dependent, stable, reversible, saturable and specific. The use of functional cAMP assays has led to the
functional classification of melatonin receptors in different tissues
throughout the body (Garcia-Perganeda
1999; Nowak et al. 1997). Since
melatonin can regulate the levels of cAMP, it could regulate the expression of
its own receptor, which is controlled by the levels of cAMP (Hazlerigg et al.
1993).
Regulation of Melatonin Receptors
Melatonin receptors can be regulated by
desensitization or downregulation.
Desensitization is a decrease in the affinity of the receptor for the
ligand, while downregulation is an internalization of the receptor. Comparison of the Kd (the
indicator of receptor affinity) or the Bmax (the
indicator of total number of receptors) before and after treatment is generally
used to differentiate between these two mechanisms. The results from this type of experiment can be misleading in the
case of melatonin since it can cross biological membranes and bind to
internalized receptors. Therefore
charged melatonin ligands (agonists or antagonists), which cannot penetrate the
membrane, are used to measure specific binding to melatonin receptors on the
surface of the cell only (Chu et al. 2002).
A daily
fluctuation in melatonin receptor mRNA and melatonin receptor protein in the
SCN and pars tuberalis (PT) has been shown (Ross et al. 1998). In PT cells, the levels of melatonin
receptor mRNA are increased following an increase in cAMP. During the daylight, when melatonin levels
are very low, there is an increase in cAMP and subsequently an increase in
melatonin receptor mRNA. During the
night when melatonin levels begin to rise, melatonin can act on its receptor
and cause a decrease in cAMP and thereby prevent any further melatonin receptor
expression. Melatonin has also been shown to cause both desensitization and
downregulation of its own receptor by regulating its phosphorylation by PKC and
PKA (Barrett et al. 1998; Ross et al. 1998).
Non-receptor
binding sites
Any response to melatonin
that occurs without meeting all the criteria described above (for classification
of melatonin receptors; see p. 9) is considered a non-receptor mediated actions
of melatonin. The following section
compares the receptor-mediated and non-receptor mediated actions of melatonin.
