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Melatonin Binding Sites

In the late 1970s 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).

Receptor-mediated Actions of Melatonin

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.

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