Polarity of TRH receptors in transfected MDCK cells is independent of endocytosis signals and G protein coupling.

Information concerning the molecular sorting of G protein-coupled receptors in polarized epithelial cells is limited. Therefore, we have expressed the receptor for thyrotropin-releasing hormone (TRH) in Madin-Darby canine kidney (MDCK) cells by adenovirus-mediated gene transfer to determine its distribution in a model cell system and to begin analyzing the molecular information responsible for its distribution. Equilibrium binding of [methyl-3H]TRH to apical and basolateral surfaces of polarized MDCK cells reveals that TRH receptors are expressed predominantly (>80%) on the basolateral cell surface.

Ectopic expression of thyrotropin releasing hormone (TRH) receptors in liver modulates organ function to regulate blood glucose by TRH.

Maintenance of blood glucose by the liver is normally initiated by extracellular regulatory molecules such as glucagon and vasopressin triggering specific hepatocyte receptors to activate the cAMP or phosphoinositide signal transduction pathways, respectively. We now show that the normal ligand-receptor regulators of blood glucose in the liver can be bypassed using an adenovirus vector expressing the mouse pituitary thyrotropin releasing hormone receptor (TRHR) cDNA ectopically in rat liver in vivo. The ectopically expressed TRHR links to the phosphoinositide pathway, providing a means to regulate liver function with TRH, an extracellular ligand that does not normally affect hepatic function.

A constitutively active mutant thyrotropin-releasing hormone receptor is chronically down-regulated in pituitary cells: evidence using chlordiazepoxide as a negative antagonist.

A carboxyl-terminus truncated mutant of the guanine nucleotide-binding (G) protein-coupled TRH receptor (TRH-R) was previously shown to exhibit constitutive, i.e. TRH-independent, activity (C335Stop TRH-R).

Thyrotropin-releasing hormone receptor activation does not elevate intracellular cyclic adenosine 3',5'-monophosphate in cells expressing high levels of receptors.

Activation of TRH receptors (TRH-R) stimulates a signal transduction pathway that leads to the formation of two second messenger molecules, inositol 1,4,5-trisphosphate and 1,2-diacylglycerol. It has been suggested that TRH may also cause an elevation of another second messenger, cAMP. As adenovirus-mediated gene transfer allows expression of TRH-R to high levels in a number of cell types, we tested again whether TRH-R activation might elevate intracellular cAMP in these more sensitive cell systems.

Expression of thyrotropin-releasing hormone receptors by adenovirus-mediated gene transfer reveals that thyrotropin-releasing hormone desensitization is cell specific.

Biological studies of seven-transmembrane region G protein-coupled receptors have been restricted by available techniques for gene transfer into mammalian cells. We have created a highly efficient adenovirus-based expression vector for the thyrotropin-releasing hormone (TRH) receptor (TRH-R), AdCMVmTRHR, to circumvent difficulties encountered when transient or stable plasmid expression systems are used. We show that infection with AdCMVmTRHR results in fully functional TRH-Rs, which can be expressed in a broad range of mammalian cell types, including those resistant to conventional transient transfection.

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.

Decreased levels of internalized thyrotropin-releasing hormone receptors after uncoupling from guanine nucleotide-binding protein and phospholipase-C.

Internalization of TRH receptor (TRH-R) is dependent on sequences/structures in the receptor carboxyl-terminal tail. Here, we studied whether coupling to guanine nucleotide-binding protein (G-protein) and phospholipase-C (PLC) is involved in internalization. We constructed two mutant TRH-Rs: delta 218-263 TRH-R, in which most of the residues that form the putative third intracellular loop were deleted, and D71A TRH-R, in which an Asp in the putative second transmembrane helix was mutated to Ala; these TRH-Rs did not activate PLC when expressed transiently in COS-1 cells.

Agonist-stimulated internalization of the thyrotropin-releasing hormone receptor is dependent on two domains in the receptor carboxyl terminus.

The thyrotropin-releasing hormone (TRH)-TRH receptor (TRHR) complex undergoes rapid transformation in cells to an acid-resistant form which appears to represent internalized agonist-receptor complex. Since residues in the carboxyl terminus of other G protein-coupled receptors appear to be involved in internalization, we studied the role of this domain in the TRHR. A mutant TRHR, C335Stop, missing the last 59 residues including 2 cysteine residues, undergoes minimal transformation to an acid-resistant form even though it binds agonist with equal affinity and activates inositol phosphate second messenger formation as effectively as wild type TRHR.

Mechanism of regulation of thyrotropin-releasing hormone receptor messenger ribonucleic acid in stably transfected rat pituitary cells.

We showed previously that the level of TRH receptor (TRH-R) mRNA in rat pituitary GH3 cells is down-regulated by TRH. Here, we study the mechanism of regulation of TRH-R mRNA in a line of GH3 cells that are stably transfected with mouse pituitary TRH-R cDNA (GH-mTRHR-1 cells). GH-mTRHR-1 cells were found to have 2.