The gamma-amino-n-butyric acid type B (GABA(B)) receptor is composed of two subunits, GABA(B)1 and GABA(B)2, belonging to the family 3 heptahelix receptors. These proteins possess two domains, a seven transmembrane core and an extracellular domain containing the agonist binding site. This binding domain is likely to fold like bacterial periplasmic binding proteins that are constituted of two lobes that close upon ligand binding. Here, using molecular modeling and site-directed mutagenesis, we have identified residues in the GABA(B)1 subunit that are critical for agonist binding and activation of the heteromeric receptor. Our data suggest that two residues (Ser(246) and Asp(471)) located within lobe I form H bonds and a salt bridge with carboxylic and amino groups of GABA, respectively, demonstrating the pivotal role of lobe I in agonist binding. Interestingly, our data also suggest that a residue within lobe II (Tyr(366)) interacts with the agonists in a closed form model of the binding domain, and its mutation into Ala converts the agonist baclofen into an antagonist. These data demonstrate the pivotal role played by the GABA(B)1 subunit in the activation of the heteromeric GABA(B) receptor and are consistent with the idea that a closed state of the binding domain of family 3 receptors is required for their activation.
Glutamate carboxypeptidase
II
(
GCPII
), also known as prostate‐specific membrane antigen (
PSMA
) or folate hydrolase, is a metallopeptidase expressed predominantly in the human brain and prostate.
GCPII
expression is considerably increased in prostate carcinoma, and the enzyme also participates in glutamate excitotoxicity in the brain. Therefore,
GCPII
represents an important diagnostic marker of prostate cancer progression and a putative target for the treatment of both prostate cancer and neuronal disorders associated with glutamate excitotoxicity. For the development of novel therapeutics, mouse models are widely used. However, although mouse
GCPII
activity has been characterized, a detailed comparison of the enzymatic activity and tissue distribution of the mouse and human
GCPII
orthologs remains lacking. In this study, we prepared extracellular mouse
GCPII
and compared it with human
GCPII
. We found that mouse
GCPII
possesses lower catalytic efficiency but similar substrate specificity compared with the human protein. Using a panel of
GCPII
inhibitors, we discovered that inhibition constants are generally similar for mouse and human
GCPII
. Furthermore, we observed highest expression of
GCPII
protein in the mouse kidney, brain, and salivary glands. Importantly, we did not detect
GCPII
in the mouse prostate. Our data suggest that the differences in enzymatic activity and inhibition profile are rather small; therefore, mouse
GCPII
can approximate human
GCPII
in drug development and testing. On the other hand, significant differences in
GCPII
tissue expression must be taken into account when developing novel
GCPII
‐based anticancer and therapeutic methods, including targeted anticancer drug delivery systems, and when using mice as a model organism.
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