Intracellular domains of the delta-subunits of Torpedo and rat acetylcholine receptors--expression, purification, and characterization.
There are quite detailed structural data on the extracellular ligand-binding domain and the intramembrane channel-forming domain of the nicotinic acetylcholine receptors (nAChR). However, the structure of the intracellular domain, which has variable amino acid sequences in different nAChR subunits, remains unknown. We expressed in Escherichia coli the intracellular loops (between transmembrane fragments TM3 and TM4) of the delta-subunits from the Torpedo californica and Rattus norvegicus muscle nAChRs. To facilitate purification, (His)6-tags were attached with or without linkers, and the effects of protein truncations at C- or N-termini were examined. The proteins were purified from inclusion bodies under denaturing conditions by Ni-NTA-chromatography. Molecular weight and peptide mass fingerprint was determined by MALDI mass spectrometry. Size-exclusion chromatography revealed that the Torpedo intracellular delta-loop refolded in an aqueous buffer was present in solution as a dimer. Phosphorylation of this protein with protein kinase A and tyrosine kinase (Abl) occurred at the same serine and tyrosine residues as in the native receptor. According to CD spectra, the secondary structure was not sensitive to phosphorylation. The rat intracellular loops could be solubilized only in the presence of non-ionic detergents or lipids. CD spectra indicate that the Torpedo and rat proteins have differences in their secondary structure. In the presence of dodecylphosphocholine, high concentrations (up to 6 mg/ml) of the Torpedo and rat intracellular loops were achieved. The results suggest that the spatial structure of the intracellular loops is dependent on environment and species, but is not changed significantly upon enzymatic phosphorylation.
Kottwitz
Denise
D
Institute of Biochemistry, Free University of Berlin, Thielallee 63, 14195 Berlin, Germany.
Kukhtina
Viktoria
V
Dergousova
Natalia
N
Alexeev
Timophey
T
Utkin
Yuri
Y
Tsetlin
Victor
V
Hucho
Ferdinand
F
D000818:Animals; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry; D015971:Gene Expression Regulation, Enzymologic; D006868:Hydrolysis; D018482:Muscle, Skeletal / Q000737:chemistry*; D010766:Phosphorylation; D017434:Protein Structure, Tertiary / Q000502:physiology; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D011505:Protein-Tyrosine Kinases / Q000737:chemistry; D051381:Rats; D011950:Receptors, Cholinergic / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D014101:Torpedo
D021122:Protein Subunits; D011950:Receptors, Cholinergic; D011993:Recombinant Fusion Proteins; D011505:Protein-Tyrosine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases