BETA-ADRENERGIC RECEPTOR STRUCTURE AND DESENSITIZATION

  • Clark, Richard B, (PI)

Project: Research projectResearch Project

Description

The overall goal of this proposal is the elucidation of the complex
mechanisms which cause the rapid desensitization and sensitization of
beta2-adrenergic receptor (beta2AR) stimulation of adenylyl cyclase (AC).
The knowledge gained will help in the understanding of diseases such as
asthma and hypertension in which these mechanisms may be altered, and in
the rationale for pharmacological
intervention. This proposal has three specific aims which are focused on determining
the role of phosphorylation/dephosphorylation in the regulation of the
responsivity of AC to beta2AR stimulation. The first aim is to determine
the precise domains of the beta2AR involved in the cAMP-dependent protein
kinase, the protein kinase C (PKC) and the beta-adrenergic receptor
kinase pathways of desensitization. beta2AR mutants will be constructed,
expressed in L cells and their regulation of AC characterized in response
to various treatments which elicit desensitization. To measure the
stoichiometry of phosphorylation, mutant beta2AR's will be constructed
containing foreign epitopes with the intent of developing a rapid high
recovery procedure for purification based on epitope
binding to affinity columns. The second aim is to assess the hypothesis that PKC-mediated
phosphorylation and inactivation of either the Gi/alpha subunits of the
GTP-binding proteins, or the catalytic subunit of AC is the mechanism of
the 4Beta-phorbol 12-myristate 13-acetate (PMA) and purinergic receptor-
induced sensitizations of AC. This will be accomplished through the use
of immunoprecipitation to measure phosphorylation of Gi/alpha subunits
and AC catalytic subunits, construction of mutants of these proteins to
aid in purification and analysis of domains involved, and reconstitution
studies to determine if Gi function can be restored. PKC isozyme
involvement in the PMA and purinergic sensitizations will be identified
by measurement of translocation by immunoprecipitations and phorbol
dibutyrate binding. The third aim is to identify phosphatases involved in the
desensitization/sensitization in intact cells through the use of
phosphatase inhibitors.
StatusFinished
Effective start/end date4/1/838/31/12

Funding

  • National Institutes of Health: $410,920.00
  • National Institutes of Health
  • National Institutes of Health: $298,000.00
  • National Institutes of Health: $134,793.00
  • National Institutes of Health
  • National Institutes of Health: $210,208.00
  • National Institutes of Health: $327,434.00
  • National Institutes of Health: $309,771.00
  • National Institutes of Health: $161,733.00
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health: $298,000.00
  • National Institutes of Health: $424,117.00
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health: $284,137.00
  • National Institutes of Health: $298,750.00
  • National Institutes of Health: $309,771.00
  • National Institutes of Health: $98,093.00
  • National Institutes of Health: $299,000.00
  • National Institutes of Health
  • National Institutes of Health: $286,067.00
  • National Institutes of Health
  • National Institutes of Health: $319,023.00

Fingerprint

Receptors, Adrenergic, beta
Adrenergic Receptors
Adenylyl Cyclases
Phosphorylation
Protein Kinases
Epinephrine
Cyclic GMP-Dependent Protein Kinases
Phosphoric Monoester Hydrolases
Alprostadil
Colforsin
Asthma
Cyclic AMP-Dependent Protein Kinases
Immunoprecipitation
Epitopes
Protein Kinase C
Gi-Go GTP-Binding Protein alpha Subunits
Catalytic Domain
Acetates
Site-Directed Mutagenesis
Hormones

Keywords

  • Medicine(all)
  • Biochemistry, Genetics and Molecular Biology(all)