TY - JOUR
T1 - The surface cyclic AMP receptor in Dictyostelium. Levels of ligand-induced phosphorylation, solubilization, identification of primary transcript, and developmental regulation of expression
AU - Klein, P.
AU - Vaughan, R.
AU - Borleis, J.
AU - Devreotes, P.
N1 - Copyright:
Copyright 2004 Elsevier B.V., All rights reserved.
PY - 1987
Y1 - 1987
N2 - A monospecific polyclonal antiserum to the surface cAMP receptor of Dictyostelium has been developed by immunization with purified receptor immobilized on particles of polyacrylamide and on nitrocellulose paper. In Western blots, the antiserum displays high affinity and specificity for both the R (M(r) 40,000) and D (M(r) 43,000) forms of the receptor previously identified by photoaffinity labeling with 8-azido-[32P]cAMP. These bands, labeled with the photoaffinity label or with 32P(i), were quantitatively and specifically immunoprecipitated, supporting co-purification data that all represent the same polypeptide. The R form, found in unstimulated cells, contained at least 0.2 mol of phosphate/mol of receptor. The D form, generated by cAMP stimulation of intact cells, contained at least 4 mol of phosphate/mol of receptor. In the absence of detergents, the receptor was exclusively located on membranes. The receptor was solubilized effectively in Triton X-100 and sedimented as a broad peak of 5-7 S on sucrose velocity gradients. Western blots of membranes isolated at different times after starvation indicate that the appearance of cell surface cAMP binding sites during the aggregation stage of development (5-6 h) is due to de novo synthesis of receptor protein. Pulse labeling with [35S]methionine indicated that the receptor is most rapidly synthesized during the preaggregation stage of development (1-3 h), prior to its maximal accumulation in membranes. The serum specifically immunoprecipitates a polypeptide of M(r) 37,000 from an in vitro translation reaction using RNA isolated from preaggregation stage cells. The time course of expression of the mRNA coding for the M(r) 37,000 polypeptide parallels the rate of receptor synthesis in vivo.
AB - A monospecific polyclonal antiserum to the surface cAMP receptor of Dictyostelium has been developed by immunization with purified receptor immobilized on particles of polyacrylamide and on nitrocellulose paper. In Western blots, the antiserum displays high affinity and specificity for both the R (M(r) 40,000) and D (M(r) 43,000) forms of the receptor previously identified by photoaffinity labeling with 8-azido-[32P]cAMP. These bands, labeled with the photoaffinity label or with 32P(i), were quantitatively and specifically immunoprecipitated, supporting co-purification data that all represent the same polypeptide. The R form, found in unstimulated cells, contained at least 0.2 mol of phosphate/mol of receptor. The D form, generated by cAMP stimulation of intact cells, contained at least 4 mol of phosphate/mol of receptor. In the absence of detergents, the receptor was exclusively located on membranes. The receptor was solubilized effectively in Triton X-100 and sedimented as a broad peak of 5-7 S on sucrose velocity gradients. Western blots of membranes isolated at different times after starvation indicate that the appearance of cell surface cAMP binding sites during the aggregation stage of development (5-6 h) is due to de novo synthesis of receptor protein. Pulse labeling with [35S]methionine indicated that the receptor is most rapidly synthesized during the preaggregation stage of development (1-3 h), prior to its maximal accumulation in membranes. The serum specifically immunoprecipitates a polypeptide of M(r) 37,000 from an in vitro translation reaction using RNA isolated from preaggregation stage cells. The time course of expression of the mRNA coding for the M(r) 37,000 polypeptide parallels the rate of receptor synthesis in vivo.
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M3 - Article
C2 - 3025212
AN - SCOPUS:0023092535
SN - 0021-9258
VL - 262
SP - 358
EP - 364
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 1
ER -