ABSTRACT
Anchoring Protein Complex Formation in Heart ............................. 148 9.1.2 Analysis of AC Interaction with Regulators and Scaffolding Proteins ...149
9.2 Issues with Classic Methods ......................................................................... 149 9.2.1 Purication on Forskolin-Agarose .................................................... 149
9.2.1.1 Advantages ......................................................................... 149 9.2.1.2 Requirements, Limitations, and Problems ......................... 150
9.2.2 Coimmunoprecipitation and Western Blot Analysis ........................ 151 9.3 Coimmunoprecipitation of AC Activity ....................................................... 151
9.3.1 Advantages ........................................................................................ 152 9.3.2 Requirements, Limitations, and Problems ....................................... 152 9.3.3 Step-by-Step Showcase Protocol ...................................................... 153
9.3.3.1 Preparation of C12E10 Detergent ......................................... 153 9.3.3.2 Preparation of Cell Lysates from Transiently
Transfected HEK293 Cells ................................................ 154 9.3.3.3 Prepare Protein A or G Beads ........................................... 154 9.3.3.4 Incubate to Form Antibody-Antigen Complexes ............... 154 9.3.3.5 Purify Protein Complexes .................................................. 155 9.3.3.6 Dowex and Alumina Chromatography .............................. 156
9.3.4 Activation of AC: Isoform-Specic Considerations ......................... 156 9.3.4.1 Purication and Activation of Gαs .................................... 157
9.3.5 Additional Considerations for Application in Tissues ...................... 157 9.3.5.1 Preparation of Lysates from Mouse Hearts ....................... 158 9.3.5.2 AC Assay ........................................................................... 158
9.3.6 Use of Disrupting Peptides to Further Dene Complexes ................ 159 9.3.6.1 IP-AC Assay with Competition Proteins (Using
Transfected HEK293 Cells) ............................................... 159
Cyclic AMP (cAMP) is a second messenger that regulates many aspects of cardiac physiology including contraction rate and force of contraction, in addition to the pathophysiology of hypertrophy and heart failure. cAMP is synthesized by adenylyl cyclase (AC), a family of nine membrane-bound enzymes that serve as nodes for cross-talk between sympathetic and parasympathetic signaling, in addition to inputs by calcium channels and other G protein-coupled receptors. In heart, cAMP activates cAMP-gated channels (HCN), cAMP-activated Rap exchange proteins (EPAC), phosphodiesterases, and most notably protein kinase A (PKA), which leads to the increased phosphorylation of numerous proteins involved in contraction.1,2 Although most ACs are readily detected in cardiac broblasts or sino-atrial node, in cardiac myocytes, the major isoforms include AC5 and AC6 and, to a lesser extent, AC4 and AC9.1,3 Deletion of either AC5 or AC6 leads to major alterations in sympathetic and parasympathetic control of ejection fraction, baroreexes, calcium handling, and stress responses.4
