María A. Diversé-Pierluissi

Tyrosine-kinase-dependent recruitment of RGS12 to the N-type calcium channel.

γ-Aminobutyric acid (GABA)B receptors couple to G o to inhibit N-type calcium channels in embryonic chick dorsal root ganglion neurons. The voltage-independent inhibition, mediated by means of a tyrosine-kinase pathway, is transient and lasts up to 100 seconds. Inhibition of endogenous RGS12, a member of the family of regulators of G-protein signalling, selectively alters the time course of voltage-independent inhibition. The RGS12 protein, in addition to the RGS domain, contains PDZ and PTB domains. Fusion proteins containing the PTB domain of RGS12 alter the rate of termination of the GABAB signal, whereas the PDZ or RGS domains of RGS12 have no observable effects. Using primary dorsal root ganglion neurons in culture, here we show an endogenous agonist-induced tyrosine-kinase-dependent complex of RGS12 and the calcium channel. These results indicate that RGS12 is a multifunctional protein capable of direct interactions through its PTB domain with the tyrosine-phosphorylated calcium channel. Recruitment of RGS proteins to G-protein effectors may represent an additional mechanism for signal termination in G-protein-coupled pathways.

β-Adrenergic receptor activation induces internalization of cardiac Cav1.2 channel complexes through a β-arrestin 1-mediated pathway

Voltage-dependent calcium channels (VDCCs) play a pivotal role in normal excitation-contraction coupling in cardiac myocytes. These channels can be modulated through activation of β-adrenergic receptors (β-ARs), which leads to an increase in calcium current (ICa-L) density through cardiac Cav1 channels as a result of phosphorylation by cAMP-dependent protein kinase A. Changes in ICa-L density and kinetics in heart failure often occur in the absence of changes in Cav1 channel expression, arguing for the importance of post-translational modification of these channels in heart disease. The precise molecular mechanisms that govern the regulation of VDCCs and their cell surface localization remain unknown. Our data show that sustained β-AR activation induces internalization of a cardiac macromolecular complex involving VDCC and β-arrestin 1 (β-Arr1) into clathrin-coated vesicles. Pretreatment of myocytes with pertussis toxin prevents the internalization of VDCCs, suggesting that Gi/o mediates this response. A peptide that selectively disrupts the interaction between CaV1.2 and β-Arr1 and tyrosine kinase inhibitors readily prevent agonist-induced VDCC internalization. These observations suggest that VDCC trafficking is mediated by G protein switching to Gi of the β-AR, which plays a prominent role in various cardiac pathologies associated with a hyperadrenergic state, such as hypertrophy and heart failure.

N-type Ca2+ Channels as Scaffold Proteins in the Assembly of Signaling Molecules for GABAB Receptor Effects

An emerging concept in signal transduction is the organization of neuronal receptors and channels into microdomains in which signaling proteins are brought together to regulate functional responses. With the multiplicity of potential protein-protein interactions arises the need for the regulation and timing of these interactions. We have identified N-type Ca2+ channel-signaling molecule complexes formed at different times upon activation of γ-aminobutyric acid, type B, receptors. The first type of interaction involves pre-association of signaling proteins such as Src kinase with the Ca2+ channel, because it is rapidly activated by the receptors and regulates the magnitude of the inhibition of the Ca2+ channel. The second type of interaction involves signaling molecules that are recruited to the channel by receptor activation and control the rate of the channel response. Recruitment of members of the Ras pathway has two effects as follows: 1) modulation of the rate of onset of the γ-aminobutyric acid-mediated inhibition of Ca2+ current, and 2) activation of MAP kinase. Our results suggest that the Ca2+ channel α1 subunit functions as a dynamic scaffold allowing assembly of intracellular signaling components that alter channel activity and route signals to the MAP kinase pathway.

Inquiry learning. Integrating content detail and critical reasoning by peer review.

Classroom lectures by experts in combination with journal clubs and Web-based discussion forums help graduate students develop critical reasoning skills.

Biochemical approaches to study interaction of calcium channels with RGS12 in primary neuronal cultures.

Regulation of the timing of voltage-dependent calcium channel activity is traditionally thought to involve heterotrimeric G-protein-signaling pathways. However, it is now being realized that these complex timing events are controlled by interactions between components of several signaling pathways and the cytoskeletal structure that serves as a scaffold element for interactions between the signaling components and the effector, the calcium channel, γ-Aminobutyric acid type B (GABA B ) receptors inhibit N-type calcium channels through Gα o -mediated activation of a tyrosine kinase pathway. Data from authors laboratory suggest that the timing of GABA-induced inhibition is a complex process that involves several signals. The α (pore-forming) subunit of the calcium channel is tyrosine phosphorylated by Src kinase. This phosphorylation makes the channel a target for the binding of a GTPase-activating protein, RGS 12. The phosphotyrosine-binding (PTB) domain of RGS12 binds to the tyrosine-phosphorylated channel, altering the kinetics of the termination of the response. This chapter describes the biochemical protocols used to determine the phosphorylation of the channel and its interaction with RGSl2.

Using Web-Based Discussion Forums as a Model of the Peer-Review Process and a Tool for Assessment

This Teaching Resource describes how to use an online asynchronous discussion as a mechanism to introduce students to the peer-review process, as well as to assess student performance and understanding. This method was applied to a graduate course on signal transduction and the Teaching Resource includes a syllabus, detailed plan for incorporating the online discussion, sample journal club questions, and sample student responses to the discussion forum, faculty responses, and student revisions.

Studies of endogenous G-protein-mediated pathways in neurons by whole-cell electrophysiology.

This chapter focuses on the study and characterization of G-protein mediated pathways that modulate voltage-dependent calcium channels in neuronal primary cultures. By monitoring the calcium current, receptor-G protein signaling events can be studied at the single-cell level with a time resolution impossible to obtain by conventional biochemical methods. The electrophysiological recording approach also allows the study of cells with minimal disruption el: the molecular environment. This is important because regulation of receptor-mediated signals depends not only on receptor subtype but also on receptor density and cellular environment. Experiments performed in heterologous systems, where the receptor is overexpressed, isolate one receptor subtype in abundance but in a foreign environment, where it may couple to signaling molecules different from its preferred interaction sites in its native environment. The regulatory properties of the receptor might also be altered as the onset and kinetics of desensitization depend on the density of receptors and signaling molecules.