This page was produced as an assignment for Genetics 677, an undergraduate course at UW-Madison.

Discussion

            To evaluate the role of GRM7 in the etiology of alcoholism in humans, this website used a wide array of publically-available genomics and bioinformatics tools to conduct an in-depth investigation into GRM7 and its encoded protein. Through review of the popular press and the scientific literature on GRM7, as well as analysis of the sequence, structure, function, expression, and evolutionary relationships of the gene, this investigation strongly suggests that GRM7 as an important factor in determining risk of alcoholism. However, as the currently available data on GRM7, which are presented here, cannot definitively address whether variation in this gene in the human population contributes to variation in susceptibility to alcoholism in the humans, further study of the gene is needed.

Conclusions

            As a first step into the investigation of GRM7, the sequence, structure, and evolutionary relationships of the gene and its encoded protein were analyzed. Located at p26.1-p25.1 on chromosome 3, GRM7 is 880,291 basepairs long and encodes five different protein isoforms, which differ in their C-termini. The predominant form of these isoforms, which is 915 amino acids long, consists of three primary domains. The first of these domains, located near the N’-terminus, is an ANF receptor domain involved in receiving glutamate. GRM7 also has a transmembrane region, near to its C-terminus, which is thought to be involved in intracellular signaling. The third domain of the GRM7 protein, which is situated in between the first two, functions as a stabilizer. Examination of the evolutionary history of this protein reveals that it has orthologs throughout the animal kingdom, including in the nematode (C. elegans), the fruit fly (D. melanogaster), and the mouse (M. musculus). Additionally, GRM7 has seven closely-related human paralogs. It is interesting to note, from the phylogenetic trees built with GRM7 and all of its homologs, that the nematode and fruit fly orthologs of GRM7 are actually more closely related to GRM7 paralogs, rather than GRM7 itself. It is also interesting to note from these analyses that, although a number of paralogs of GRM7 exist, its orthologs not only have not been lost over evolutionary time in other animals, but they have retained considerable sequence similarity. The analysis of the sequence, structure, and evolutionary relationships of GRM7 suggests that this protein plays a unique, but critical, role in the normal functioning of glutamate neurotransmission. Additionally, the similarities and differences between GRM7 and its various homologs suggest that mouse is likely to be the most informative model organism for studying GRM7, followed by fruit fly, and lastly, nematode. While currently the zebrafish (Danio rerio) GRM7 ortholog is only a predicted protein, study of GRM7 in this model organism is also likely to be informative in the future.
             In the next step of the investigation conducted for this website, GRM7 and its homologs were used to determine the function of GRM7. The Gene Ontology of human GRM7 showed that, consistent with its classification as a class III metabotropic glutamate receptor, it is found integral to the plasma membrane and is involved in negative regulation of adenylate cyclase activity, as well as synaptic transmission. Interestingly, GRM7 functions in both the pre- and postsynapase, suggesting it can act as a glutamate sensor for neurons when they are sending as well as receiving a signal. While the gene ontology provided important information about the molecular function of GRM7, analysis of the mutant phenotypes produced by knock-down of GRM7 orthologs in other organisms provided insight into the biological roles of this protein. RNAi knock-down of the nematode GRM7 ortholog, mgl-1, showed that GRM7 is likely to play a role in regulating growth. Additionally, the phenotype produced by RNAi knock-down of the fruit fly GRM7 ortholog, mGluRA, showed that GRM7 may play a role in dictating synapse morphology. This finding was interesting in that it suggests that mutations in GRM7 in humans could produce altered synapse morphology. Perhaps, in turn, this could produce altered response to alcohol, and an altered risk of developing alcoholism. While the fruit fly and nematode mutant phenotypes were informative, phenotypes produced by knocking out the mouse ortholog of human GRM7, Grm7, were the most interesting, since this ortholog shows the highest similarity of all model organism orthologs to human GRM7. Mice homologous for a knock-out of Grm7 showed several defects, primarily in processes involving learning and memory. These findings point to the centrality of GRM7 in a large number of processes important to normal brain functioning, and suggest that treating alcoholism through changing the functioning of the GRM7 may cause a number of unintended and, potentially, unwanted side-effects. Perhaps, a more successful approach, when our understanding of the role of GRM7 in alcoholism is better, will be in prevention rather than treatment: people identified as carrying mutations in GRM7 that confer increased susceptibility to alcoholism can be advised to avoid behaviors and environments that tend to lead to the disease.
            While analysis of the function of GRM7 gave important insights into the general role of GRM7 in the human brain, analysis of the expression of GRM7 provided a framework for assessing the role of this gene in the etiology of alcoholism. Expression levels of GRM7 were of exceptional importance in evaluating the contributions of this gene to alcoholism in humans because, as discussed in the popular press and scientific reviews on this website, the link between GRM7 and alcoholism was originally identified through the finding that mice with lowered Grm7 expression levels exhibited increased alcohol preference. Additional evidence that levels of GRM7 expression contribute to alcoholism was provided by a chemical genetics study showing that an agonist that selectively increases GRM7 activity decreases alcohol preference in mice. These studies imply that the levels of GRM7 activity play a central role in determining an individual’s preference for alcohol, and therefore, their likelihood of developing alcoholism. If GRM7 does, in fact, contribute to risk of alcoholism in humans, it is likely due to genetic variation that leads to variation in levels of GRM7 expression. While the investigations conducted for this website revealed that studies of GRM7 expression levels in humans with relation to alcohol use have not yet been performed, an interesting MicroArray study comparing levels of GRM7 expression in the hippocampi of males and females revealed that human males, like the alcohol-preferring mice discussed above, have lower GRM7 expression. As males are known to have higher rates of alcohol dependence than females, this study, taken in light of the mouse study linking Grm7 to alcohol preference, suggests that differing levels of GRM7 expression between the sexes may be playing a role in their different rates of alcoholism. Additionally, this study, as well as the chemical genetics study mentioned above, imply that susceptibility to alcoholism may not only be due to genomic variation causing lowered gene expression levels, but also to factors interacting with GRM7 that cause it to be less active. The protein network for GRM7 revealed a number of predicted regulatory interaction partners that could influence GRM7 activity levels. Certainly, based on the many predicted post-translational modification sites for GRM7, the activity of the protein has the potential to be highly-regulated. These analyses of GRM7 expression levels suggest that genetic variation that results in lowered GRM7 activity, either through mutations in GRM7 itself or through mutations in one of the genes encoding a protein that regulates GRM7 activity, could contribute to variation of risk of alcoholism in humans. However, it remains to be seen whether this genetic variation truly exists in human populations.

Future Directions

            As it is still unknown whether GRM7 differs in alcoholics and non-alcoholics, further research into the role of GRM7 in alcoholism should focus on firstly, exploring whether a difference exists in human alcoholics and non-alcoholics in gene expression levels, and secondly, validating GRM7’s protein interaction network. One way to determine whether GRM7 expression levels differ in alcoholics and non-alcoholics would be to perform a MicroArray study examining the levels of GRM7 mRNA in post-mortem human brain samples (particularly in the hippocampus, cerebral cortex and cerebellum, since GRM7 expression is known to be highest in these brain structures) of alcoholics and non-alcoholics. If lowered expression of GRM7 mRNA was found in alcoholics, it would indicate that GRM7 probably contributes to alcoholism in humans in the same way it contributes to alcohol preference in mice. If this were true, the next step that could be taken would be to map the location of the variant causing differed levels of GRM7 expression. Similarly to in mice, this variant could be in a regulatory region of GRM7, or perhaps, it could also be in a gene encoding a regulator of GRM7 gene expression. However, in this second case, variation GRM7 itself would not be the primary cause of alcoholism risk, but rather variation in the regulator gene of GRM7 expression would be. If differences in GRM7 mRNA levels were not found to differ, it may also be possible that the relative levels of GRM7 protein in the brain contribute to susceptibility to alcoholism, and therefore, that, like in the case just discussed, the GRM7 gene contributes indirectly to risk of alcoholism. If variation in the GRM7 activity levels, whether caused by variation in GRM7 itself or by in variation in an interactor, contribute to alcoholism, it would be desirable to develop a drug that can help alcoholics overcome their addiction. However, as has been shown by both knock-out and chemical genetics studies in mice, direct manipulation of GRM7 creates a number of undesirable effects, in addition to the wanted effect of reduced alcohol preference. A possible alternative to direct manipulation of GRM7 would be to control GRM7 activity level through manipulation of the activity of one of its regulators. As many of the interactions found for GRM7 in its protein interaction network were only predicted, an important first step in development  of such a drug would be to verify the many predicted protein interactions for GRM7, as well as identify any new interactors that have previously been missed. One potential method to do this would be to perform a yeast two-hybrid experiment, screening GRM7 against other proteins expressed in the brain. Due to the high false positive rate of interactions identified with the two-hybrid experiment, interactions identified by this method could be confirmed using immunoprecipitation followed by mass spectrometry. Exploration of how GRM7 expression levels differ in humans, as well as verification of the protein interaction network for this gene are likely to provide important insights into the role of GRM7 in alcoholism as well as suggest possible new methods of treatment for the disease.


Jennifer Wagner
wagner4@wisc.edu
Updated May 14, 2009
http://www.gen677.weebly.com