Overexpression of the cAMP receptor 1 in growing Dictyostelium cells.
cAR1, the cAMP receptor expressed normally during the early aggregation stage of the Dictyostelium developmental program, has been expressed during the growth stage, when only low amounts of endogenous receptors are present. Transformants expressing cAR1 have 7-40 times over growth stage and 3-5-fold over aggregation stage levels of endogenous receptors. The high amounts of cAR1 protein expressed constitutively throughout early development did not drastically disrupt the developmental program; the onset of aggregation was delayed by 1-3 h, and then subsequent stages proceeded normally. The affinity of the expressed cAR1 was similar to that of the endogenous receptors in aggregation stage cells when measured either in phosphate buffer (two affinity states with Kd's of approximately 30 and 300 nM) or in 3 M ammonium sulfate (one affinity state with a Kd of 2-3 nM). When expressed during growth, cAR1 did not appear to couple to its normal effectors since these cells failed to carry out chemotaxis or to elevate cGMP or cAMP levels when stimulated with cAMP. However, cAMP stimulated phosphorylation, and loss of ligand binding of cAR1 did occur. Like aggregation stage control cells, the cAR1 protein shifted in apparent molecular mass from 40 to 43 kDa and became highly phosphorylated when exposed to cAMP. In addition, the number of surface cAMP binding sites in cAR1 cells was reduced by over 80% during prolonged cAMP stimulation. These results define a useful system to express altered cAR1 proteins and examine their regulatory functions.
Johnson
R L
RL
Department of Biological Chemistry, School of Medicine, Johns Hopkins University, Baltimore, Maryland 21205.
Vaughan
R A
RA
Caterina
M J
MJ
Van Haastert
P J
PJ
Devreotes
P N
PN
D000242:Cyclic AMP / Q000378:metabolism; D004023:Dictyostelium / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D011953:Receptors, Cyclic AMP / Q000235:genetics* / Q000502:physiology
D005656:Fungal Proteins; D011953:Receptors, Cyclic AMP; D000242:Cyclic AMP