G Protein Activation by Human Dopamine D3 Receptors by Cussac, Newman-Tancredi, Pasteau, and Millan - Essay Example

Compared to D2, D3 receptors less effectively bind with pertussis-sensitive Gi/Go proteins, suggesting that the pathways of D2 and D3 in promoting mitogenesis are not entirely the same, and D3-mediated growth may not even involve MAPK. However, if the contrary is true, effective external control of mitogenesis, such as that being done in cancer therapy, cannot be attained by modulating steps of the D2 pathway alone. It can thus be agreed upon that the D3 pathway warranted investigation.

Comments on the methods

The researchers thus wanted to determine 1) the effect of D3 ligands on the phosphorylation state of MAPK, and2) the signal transduction factors involved in this stimulation. To do this, D3-expressing Chinese hamster ovary (CHO) cells were exposed to different substances which inhibit proposed key factors in MAPK activation. The use of CHO is probably due to the fact that it is mammalian (and thus closely similar to that of man) and it can be inserted with genes from other organisms to determine their function. Currently, however, human cell lines that can be transfected are already commercially available, and they could have been used in this study in order for their results to be more translatable to human function.

The substances used were summarized in table 1. A range of concentrations could have been used, instead of only one, since substances may not be able to function if applied in amounts less than their effective concentration.

Table 1. Inhibitory substances used to determine signal transduction factors in D3-mediated MAPK activation

Activity

Substances used

D3 stimulant

100 nM dopamine (DA), at which maximal MAPK  phosphorylation was observed

20 ng/ml fibroblast growth factor (FGF)

Kinase inhibitors

PI-3 kinase inhibitors: 1 - 10 µM Wortmannin, 30 µM LY 294002

Protein tyrosine kinase (PTK) inhibitors: genistein, lavendustin A

MAPK kinase (MEK) inhibitor: 50 µM PD 98059

Protein kinase C (PKC) inhibitors

1 mM phorbol-12-myristate-

13-acetate (PMA)

10 µM Ro 31-8220

Go 6976

Go 6983

Gi/Go inhibitor

100 ng/ml Pertussis toxin (PTX)

D3 agonists

 100 nM (+)7-OH-DPAT

100 nM PD 128,907

DA antagonists

Haloperidol

GR218,231

S14297

   After this, activation of MAPK was revealed using a monoclonal antibody specific for phosphorylated and unphosphorylated forms of MAPK. A secondary antibody with horseradish peroxidase (HRP) was also used for chemiluminescence. This is a standard immunoblotting technique still being used now. It was commendable that immunoblots were repeated at least three times.

Comments on the data

Dopamine was able to phosphorylate both MAPK, with that of pp42 stronger than the other, and the amount phosphorylated did not wane for at least 20 min. DA was then used in the subsequent experiments. Dopaminergic antagonists also attenuated DA-induced MAPK activation, with S14297 less than the others, further establishing the MAPK-activating abilities of D3.  S14297 was also found to have partial MAPK stimulatory activity.

In D3-containing CHO exposed to DA, pretreatment of PTX blocked DA- and D3 agonist-mediated MAPK phosphorylation, indicating that the pathway between D3 and MAPK implicated Gi/Go proteins. In contrast, all kinase inhibitors, except PD 98059, did not affect D3 and MAPK interaction.

The researchers also did overnight exposure of CHO to PMA to supposedly decrease PKC. With or without PKC, MAPK did not seem to be activated without DA, and this was confirmed when DA was added and subsequent phosphorylation of MAPK was observed. This indicated a PKC-independent, DA-mediated MAPK activation. However, an unexpected finding was found when, in the presence of PKC and absence of DA, MAPK was produced.

The investigators also used other PKC inhibitors, in case PKC was resistant to PMA. Using Ro 31-8220, MAPK was phosphorylated either at supposedly low or high levels of PKC, even without DA. On the other hand, Go 6976 did not affect DA activation by MAPK, while large amounts of Go 6983 prevented MAPK phosphorylation even in the presence of DA.

Based on these results, DA-mediated MAPK activation involves Gi/Go proteins, MEK, and Go 6983-sensitive PKC, and this analysis of immunoblots confirms these findings. However, according to the researchers, PKCs involved in DA-mediated MAPK phosphorylation are also Ro 31-8220-sensitive but PMA-resistant. This is true if only immunoblots with DA are observed. However, it fails to explain why at some wells without DA or even PKC (supposedly), phosphorylated MAPK was still observed. This means that a MAPK-activating process independent of DA, and possibly D3, must be present during the experiment, confounding the results of the study.