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Fuller M.D.,University of Washington | Emrick M.A.,University of Washington | Emrick M.A.,VLST Corporation | Sadilek M.,University of Washington | And 2 more authors.
Science Signaling | Year: 2010

During the fight-or-flight response, the sympathetic nervous system stimulates L-type calcium ion (Ca2+) currents conducted by Ca V1 channels through activation of β-adrenergic receptors, adenylyl cyclase, and phosphorylation by adenosine 3′,5′- monophosphate-dependent protein kinase [also known as protein kinase A (PKA)], increasing contractility of skeletal and cardiac muscles. We reconstituted this regulation of cardiac CaV1.2 channels in non-muscle cells by forming an autoinhibitory signaling complex composed of CaV1.2Δ1800 (a form of the channel truncated at the in vivo site of proteolytic processing), its noncovalently associated distal carboxyl-terminal domain, the auxiliary α2δ1 and β2b subunits, and A-kinase anchoring protein 15 (AKAP15). A factor of 3.6 range of Ca V1.2 channel activity was observed from a minimumin the presence of protein kinase inhibitors to a maximum upon activation of adenylyl cyclase. Basal CaV1.2 channel activity in unstimulated cells was regulated by phosphorylation of serine-1700 and threonine-1704, two residues located at the interface between the distal and the proximal carboxyl-terminal regulatory domains, whereas further stimulation of channel activity through the PKA signaling pathway only required phosphorylation of serine-1700. Our results define a conceptual framework for CaV1.2 channel regulation and identify sites of phosphorylation that regulate channel activity.

Bauman A.T.,Oregon Health And Science University | Bauman A.T.,VLST Corporation | Broers B.A.,Oregon Health And Science University | Kline C.D.,Oregon Health And Science University | Blackburn N.J.,Oregon Health And Science University
Biochemistry | Year: 2011

The pH dependence of native peptidylglycine monooxygenase (PHM) and its M314H variant has been studied in detail. For wild-type (WT) PHM, the intensity of the Cu-S interaction visible in the Cu(I) extended X-ray absorption fine structure (EXAFS) data is inversely proportional to catalytic activity over the pH range of 3-8. A previous model based on more limited data was interpreted in terms of two protein conformations involving an inactive Met-on form and an active flexible Met-off form [Bauman, A. T., et al. (2006) Biochemistry 45, 11140-11150] that derived its catalytic activity from the ability to couple into vibrational modes critical for proton tunneling. The new studies comparing the WT and M314H variant have led to the evolution of this model, in which the Met-on form has been found to be derived from coordination of an additional Met residue, rather than a more rigid conformer of M314 as previously proposed. The catalytic activity of the mutant decreased by 96% because of effects on both k cat and K M, but it displayed the same activity-pH profile with a maximum around pH 6. At pH 8, the reduced Cu(I) form gave spectra that could be simulated by replacement of the Cu M Cu-S(Met) interaction with a Cu-N/O interaction, but the data did not unambiguously assign the ligand to the imidazole side chain of H314. At pH 3.5, the EXAFS still showed the presence of a strong Cu-S interaction, establishing that the Met-on form observed at low pH in WT cannot be due to a strengthening of the Cu M-methionine interaction but must arise from a different Cu-S interaction. Therefore, lowering the pH causes a conformational change at one of the Cu centers that brings a new S donor residue into a favorable orientation for coordination to copper and generates an inactive form. Cys coordination is unlikely because all Cys residues in PHM are engaged in disulfide cross-links. Sequence comparison with the PHM homologues tyramine β-monooxygenase and dopamine β-monooxygenase suggests that M109 (adjacent to H site ligands H107 and H108) is the most likely candidate. A model is presented in which H108 is protonated with a pK a of 4.6 to generate the inactive low-pH form with Cu H coordinated by M109, H107, and H172. © 2011 American Chemical Society.

VLST Corporation | Date: 2010-08-27

An antikine antibody binds to two, three, four, five or more CC chemokines, such as RANTES/CCL5, MIP-1/CCL3, MIP-1/CCL4, or MCP-1/CCL2. Methods for affinity maturation and humanization of antikine antibodies as well as the production of hybridoma cell lines producing antikine antibodies by sequential immunization are also disclosed.

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