THYROTROPIN-RELEASING-HORMONE (TRH) RECEPTOR NUMBER DETERMINES THE SIZE OF THE TRH-RESPONSIVE PHOSPHOINOSITIDE POOL - DEMONSTRATION USING CONTROLLED EXPRESSION OF TRH RECEPTORS BY ADENOVIRUS-MEDIATED GENE-TRANSFER

We use an adenovirus vector, AdCMVmTRHR, to express thyrotropin-releas ing hormone (TRH) receptors (TRH-Rs) to determine whether the size of the hormone-responsive phosphoinositide pool in mammalian cells is dir ectly related to receptor number. Infection of HeLa cells with increas ing numbers of AdCMVmTRHR caused time-dependent graded expression of T RH-Rs. Measurement of cytoplasmic free Ca2+ in individual cells permit ted quantitation of the fraction of cells responsive to TRH. Infection with 100 AdCMVmTRHR particles/cell or more led to TRH responsiveness in greater than or equal to 90% of HeLa cells. Measurement of prelabel ed phosphoinositides hydrolyzed during prolonged TRH stimulation asses ses the size of the TRH-responsive pool. In cells infected with AdCMVm TRHR for 24 h, the size of the TRH-responsive phosphoinositide pool in creased with increasing TRH-R expression. The TRH-responsive pool also increased with time after infection as the number of TRH-Rs increased , Similar observations were made in GHY and KB cells. These data confi rm our previous suggestion (Cubitt, A. B., Geras-Raaka, E., and Gershe ngorn, M. C. (1990) Biochem. J, 271, 331-336) that there are hormone-r esponsive and -unresponsive pools of cellular phosphoinositides and th at the maximal size of the TRH-responsive pool is directly related to the number of TRH-Rs.