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-releasing hormone (TRH) receptors (TRH-Rs) to determine whether the size of the hormone-responsive phosphoinositide pool in mammalian cells is directly related to receptor number. Infection of HeLa cells with increasing numbers of AdCMVmTRHR caused time-dependent graded expression of TRH-Rs. Measurement of cytoplasmic free Ca2+ in individual cells permitted quantitation of the fraction of cells responsive to TRH. Infection with 100 AdCMVmTRHR particles/cell or more led to TRH responsiveness in > or = 90% of HeLa cells. Measurement of prelabeled phosphoinositides hydrolyzed during prolonged TRH stimulation assesses the size of the TRH-responsive pool. In cells infected with AdCMVmTRHR for 24 h, the size of the TRH-responsive phosphoinositide pool increased 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 confirm our previous suggestion (Cubitt, A. B., Geras-Raaka, E., and Gershengorn, M. C. (1990) Biochem. J. 271, 331-336) that there are hormone-responsive and -unresponsive pools of cellular phosphoinositides and that the maximal size of the TRH-responsive pool is directly related to the number of TRH-Rs.
Gershengorn
M C
MC
Department of Medicine, Cornell University Medical College, New York, New York 10021.
Heinflink
M
M
Nussenzveig
D R
DR
Hinkle
P M
PM
Falck-Pedersen
E
E
D000256:Adenoviridae; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D005260:Female; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D007624:KB Cells; D007700:Kinetics; D008322:Mammals; D010716:Phosphatidylinositols / Q000378:metabolism*; D018025:Receptors, Thyrotropin-Releasing Hormone / Q000096:biosynthesis* / Q000502:physiology*; D013973:Thyrotropin-Releasing Hormone / Q000494:pharmacology*; D013997:Time Factors; D002583:Uterine Cervical Neoplasms
D010716:Phosphatidylinositols; D018025:Receptors, Thyrotropin-Releasing Hormone; D013973:Thyrotropin-Releasing Hormone; D002118:Calcium