Glucose-induced cAMP signalling in yeast requires both a G-protein coupledreceptor system for extracellular glucose detection and a separable hexosekinase-dependent sensing process

In Saccharomyces cerevisiae, glucose activation of cAMP synthesis requires both the presence of the G-protein-coupled receptor (GPCR) system, Gpr1-Gpa 2, and uptake and phosphorylation of the sugar. In a hxt-null strain that l acks all physiologically important glucose carriers, glucose transport as w ell as glucose-induced cAMP signalling can be restored by constitutive expr ession of the galactose permease. Hence, the glucose transporters do not se em to have a regulatory function but are only required for glucose uptake. We established a system in which the GPCR-dependent glucose-sensing process is separated from the glucose phosphorylation process. It is based on the specific transport and hydrolysis of maltose providing intracellular glucos e in the absence of glucose transport. Preaddition of a low concentration ( 0.7 mM) of maltose to derepressed hxt-null cells and subsequent addition of glucose restored the glucose-induced cAMP signalling, although there was n o glucose uptake. Addition of a low concentration of maltose itself does no t increase the cAMP level but enhances Glu6P and apparently fulfils the int racellular glucose phosphorylation requirement for activation of the cAMP p athway by extracellular glucose. This system enabled us to analyse the affi nity and specificity of the GPCR system for fermentable sugars. Gpr1 displa yed a very low affinity for glucose (apparent K-a = 75 mM) and responded sp ecifically to extracellular alpha and beta D-glucose and sucrose, but not t o fructose, mannose or any glucose analogues tested. The presence of the co nstitutively active Gpa2(val132) allele in a wild-type strain bypassed the requirement for Gpr1 and increased the low cAMP signal induced by fructose and by low glucose up to the same intensity as the high glucose signal. The refore, the low cAMP increases observed with fructose and low glucose in wi ld-type cells result only from the low sensitivity of the Gpr1-Gpa2 system and not from the intracellular sugar kinase-dependent process. In conclusio n, we have shown that the two essential requirements for glucose-induced ac tivation of cAMP synthesis can be fulfilled separately: an extracellular gl ucose detection process dependent on Gpr1 and an intracellular sugar-sensin g process requiring the hexose kinases.