Point Mutation In Transmembrane Domain Research Articles

Probing the Metabotropic Glutamate Receptor 5 (mGlu5) Positive Allosteric Modulator (PAM) Binding Pocket: Discovery of Point Mutations That Engender a “Molecular Switch” in PAM Pharmacology

Positive allosteric modulation of metabotropic glutamate receptor subtype 5 (mGlu₅) is a promising novel approach for the treatment of schizophrenia and cognitive disorders. Allosteric binding sites are topographically distinct from the endogenous ligand (orthosteric) binding site, allowing for co-occupation of a single receptor with the endogenous ligand and an allosteric modulator. Negative allosteric modulators (NAMs) inhibit and positive allosteric modulators (PAMs) enhance the affinity and/or efficacy of the orthosteric agonist. The molecular determinants that govern mGlu₅ modulator affinity versus cooperativity are not well understood. Focusing on the modulators based on the acetylene scaffold, we sought to determine the molecular interactions that contribute to PAM versus NAM pharmacology. Generation of a comparative model of the transmembrane-spanning region of mGlu₅ served as a tool to predict and interpret the impact of mutations in this region. Application of an operational model of allosterism allowed for determination of PAM and NAM affinity estimates at receptor constructs that possessed no detectable radioligand binding as well as delineation of effects on affinity versus cooperativity. Novel mutations within the transmembrane domain (TM) regions were identified that had differential effects on acetylene PAMs versus 2-methyl-6-(phenylethynyl)-pyridine, a prototypical NAM. Three conserved amino acids (Y658, T780, and S808) and two nonconserved residues (P654 and A809) were identified as key determinants of PAM activity. Interestingly, we identified two point mutations in TMs 6 and 7 that, when mutated, engender a mode switch in the pharmacology of certain PAMs.

Hydrophobic anions potently and uncompetitively antagonize GABAA receptor function in the absence of a conventional binding site

A 'lock-and-key' binding site typically accounts for the effect of receptor antagonists. However, sulphated neurosteroids are potent non-competitive antagonists of GABA(A) receptors without a clear structure-activity relationship. To gain new insights, we tested two structurally unrelated hydrophobic anions with superficially similar properties to sulphated neurosteroids. We used voltage-clamp techniques in Xenopus oocytes and hippocampal neurons to characterize dipicrylamine (DPA) and tetraphenylborate (TPB), compounds previously used to probe membrane structure and voltage-gated ion channel function. Both DPA and TPB potently antagonized GABA(A) receptors. DPA exhibited an IC₅₀ near 60 nM at half-maximal GABA concentration and antagonism with features indistinguishable from pregnenolone sulphate antagonism, including sensitivity to a point mutation in transmembrane domain 2 of the α1 subunit. Bovine serum albumin, which scavenges free membrane-associated DPA, accelerated both capacitance offset and antagonism washout. Membrane interactions and antagonism were explored using the voltage-dependent movement of DPA between membrane leaflets. Washout of DPA antagonism was strongly voltage-dependent, paralleling DPA membrane loss, although steady-state antagonism lacked voltage dependence. At antagonist concentrations, DPA failed to affect inhibitory post-synaptic current (IPSC) amplitude or decay, but DPA accelerated pharmacologically prolonged IPSCs. Neurosteroid-like GABA(A) receptor antagonism appears to lacks a conventional binding site. These features highlight key roles of membrane interactions in antagonism. Because its membrane mobility can be controlled, DPA may be a useful probe of GABA(A) receptors, but its effects on excitability via GABA(A) receptors raise caveats for its use in monitoring neuronal activity.

SHC and GRB-2 Are Constitutively Activated by an Epidermal Growth Factor Receptor with a Point Mutation in the Transmembrane Domain

A single point mutation, Glu627--> Val, equivalent to the activating mutation in the Neu oncogene, was inserted in the transmembrane domain of the human epidermal growth factor (EGF) receptor. Unlike the wild type, Glu627-EGF receptor, transfected in NIH3T3 cells, gave rise to focal transformation and growth in agar even in the absence EGF. Constitutive activity of mutant EGF receptor amounted to 20% of that of wild type receptor stimulated by EGF. In addition, the mutant receptor was more sensitive to EGF, reaching maximum transforming activity at 5 ng/ml EGF. NIH3T3 cells expressing Glu627-EGF receptor showed a transformed phenotype and were not arrested in G0 upon serum deprivation. The mutant receptor was constitutively autophosphorylated, and several other cellular proteins were phosphorylated on tyrosine in absence of the ligand. Among these, the SHC adaptor protein was phosphorylated in absence of EGF, the other adaptor, GRB-2 was constitutively associated with the Glu627-EGF receptor in vivo and in vitro, and mitogen-activated protein kinase was constitutively phosphorylated. In contrast, other EGF receptor substrates, like phospholipase C gamma, were not phosphorylated in absence of EGF. The mutant receptor showed a higher sensitivity to cleavage by calpain both in absence and presence of EGF, appeared as a 170- and 150-kDa doublet in cell extracts, and a specific calpain inhibitor blocked the appearance of the 150-kDa form. Since the calpain cleavage site is located in the receptor cytoplasmic tail, this finding suggests that the Glu627 mutation induces a slightly different conformation in the EGF receptor intracellular domain. In conclusion, our data show that a point mutation in the EGF receptor transmembrane domain was able to constitutively activate the receptor and to induce transformation via constitutive activation of the Ras pathway.