Extracellular cAMP serves as a primary signaling molecule to regulate the development of Dictyostelium discoideum. It is required for chemotaxis, aggregation, cytodifferentiation, and morphogenetic movement. The receptors for cAMP are members of the family of cell-surface receptors that are linked to G proteins and characterized by seven putative transmembrane domains. Previously, we have isolated the gene for the cAMP receptor subtype 1 (CAR1) from Dictyostelium and suggested that several genes related to CAR1 were present in the genome. Here, we describe a family of cAMP receptor genes of Dictyostelium and the isolation and function of the gene for the cAMP receptor subtype 2, CAR2. CAR2 is structurally similar to CAR1. Overall, their transmembrane and loop domains are -75% identical in amino acid sequence; however, their carboxyl termini are quite dissimilar; CAR2 possesses homopolymeric runs of histidines and asparagines that are absent from the corresponding region in CAR1. Although CAR1 is maximally expressed during the early stages of development, CAR2 is expressed only after cells have aggregated and, then, preferentially in prestalk cells. Transgenic Dictyostelium that have had their wild-type CAR2 gene replaced by a defective copy using homologous recombination proceed through early development but are detained at the tight mound stage. CAR2 may be required for cAMP-directed sorting of prestalk cells during pattern formation within the aggregation mound. Furthermore, although prestalk genes are expressed normally in aggregates that lack CAR2, they exhibit an enhanced expression of prespore-specific mRNA. Previously, we had shown that there was a requirement for CAR1 during early development. The present results demonstrate that the multiple responses of Dictyostelium to cAMP are regulated by distinct cAMP receptors that are encoded by unique genes.
Pseudoplasmodia of developing Dictyostelium are organized with anteroposterior polarity. We have isolated CAR4, the gene for a new cell-surface, G protein-linked cAMP receptor. CAR4 mRNA is initially expressed during tip elongation and continues to accumulate into culmination. CAR4 is maximally expressed in pseudoplasmodia anteriors which are centers for extracellular cAMP signaling and for organization of cellular patterning. Although car4 null cells progress unperturbed through early development, they exhibit major patterning aberrations as the anteroposterior axis becomes established. Prestalk gene expression is significantly reduced in car4 nulls, whereas prespore-specific markers are overexpressed and detected in zones normally restricted to prestalk cells. Patterning defects are similarly apparent in terminally differentiated fruiting bodies. Our results show that cAMP signaling is required for pattern formation and cellular differentiation during late Dictyostelium development.
Dictyostelium discoideum has a well characterized life cycle where unicellular growth and multicellular development are separated events. Development is dependent upon signal transduction mediated by cell surface, cAMP receptor/G protein linkages. Secreted cAMP acts extracellularly as a primary signal and chemoattractant. There are 4 genes for the distinct cAMP receptor subtypes, CAR1, CAR2, CAR3 and CAR4. These subtypes are expressed with temporally and spatially specific patterns and cells carrying null mutations for each gene have distinct developmental phenotypes. These results indicate an essential role for cAMP signalling throughout Dictyostelium development to regulate such diverse pathways as cell motility, aggregation (multicellularity), cytodifferentiation, pattern formation and cell type-specific gene expression.
We have previously shown that several genes expressed during Dictyostelium development could be induced in shaking culture by exogenous cAMP, even though the accumulation of intracellular cAMP was inhibited.
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