Abstract
Seven-membrane span receptors transduce a wide range of signals across the plasma membrane. One member of this family, the cAMP receptor, cAR1, of Dictyostelium, mediates some responses (e.g. adenylyl cyclase activation, multicellular aggregation) which require G-proteins and others (e.g. Ca2+ influx, loss of ligand binding, cAR1 phosphorylation) which appear to be G-protein-independent. In this study, we randomly mutagenized the NH2-terminal eight amino acids of the third intracellular loop of cAR1 and examined the ability of these mutants to exhibit the three G-protein-independent responses listed above. Most mutants (classes I, II) exhibited wild-type or midly defective responses. Several mutants (class III), however, were severely impaired in all three processes but not in cAMP binding. Furthermore, these mutants failed to couple productively with G-proteins and could not replace cAR1 in a car1- cell. For these reasons, we propose that class III mutations interfere with the formation of an "active" conformation of the receptor.
Highlights
Seven-membranespanreceptorstransduce a wide signaling, andgene expression changes which occur within the range of signals across the plasma membOrnaenme.em- initial hours of development
We examined the role of the CAR1 third intracellular loop in cAMP-mediated activation and desensitization events by randomly mutagenizing a portion of this domain
Mu- telium amoebae leads to a rapid reduction of up to 80% of tant A42 is noteworthy since it possesses a single amino acid surface CAMP-bindingsites within7 min[12,13,38]
Summary
Cells and Cell Culture-AX-3 cells were grownin shaking culture in HW [32]. Transformed cell lines were maintained on Petri dishes in HL5 with G418 (20 pglml, Sigma) or in HL5 alone (JB-4 cells only). A 3.7-kb CZaI fragment frompDU3B1 [33] was subcloned betweenthese inserts to create pMC25 This vector was linearized and transformed into DH-1 cellsby electroporation,and uracil prototrophs were selected clonally. Nonspecific uptake was assessed by adding IO4 M unlabeled CAMPto parallel samples.At the indicated times, cells were diluted 15-foldin ice-cold PB, lo M CAMP, immediately pelleted (5min,4000 rpm, HS-4 rotor), and washed three more times in PB. Cells were lysed through 5-pm filters (Millipore)and the lysates pelleted (5 min, 10,000rpm, SS34 rotor).The pellets were resuspended in PB to a density of 6 x lo7cell equivalentdml and kept on ice.Binding cARl Third Loop Random Mutagenesis of 2 n~ t3H1cAMPbinding was assessed by spinning through sili- wild-typecAR1-transformed cells bind 15-20-fold as much cone oil as described [50]
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