Inefficient maturation of the rat luteinizing hormone receptor. A putative way to regulate receptor numbers at the cell surface.

Increasing evidence suggests that the folding and maturation of monomeric proteins and assembly of multimeric protein complexes in the endoplasmic reticulum (ER) may be inefficient not only for mutants that carry changes in the primary structure but also for wild type proteins. In the present study, we demonstrate that the rat luteinizing hormone receptor, a G protein-coupled receptor, is one of these proteins that matures inefficiently and appears to be very prone to premature degradation. A substantial portion of the receptors in stably transfected human embryonic kidney 293 cells existed in immature form of M(r) 73,000, containing high mannose-type N-linked glycans. In metabolic pulse-chase studies, only approximately 20% of these receptor precursors were found to gain hormone binding ability and matured to a form of M(r) 90,000, containing bi- and multiantennary sialylated N-linked glycans. The rest had a propensity to form disulfide-bonded complexes with a M(r) 120,000 protein in the ER membrane and were eventually targeted for degradation in proteasomes. The number of membrane-bound receptor precursors increased when proteasomal degradation was inhibited, and no cytosolic receptor forms were detected, suggesting that retrotranslocation of the misfolded/incompletely folded receptors is tightly coupled to proteasomal function. Furthermore, a proteasomal blockade was found to increase the number of receptors that were capable of hormone binding. Thus, these results raise the interesting possibility that luteinizing hormone receptor expression at the cell surface may be controlled at the ER level by regulating the number of newly synthesized proteins that will mature and escape the ER quality control and premature degradation.

Expression of the mature luteinizing hormone receptor in rodent urogenital and adrenal tissues is developmentally regulated at a posttranslational level.

The LH receptor (LHR) is a G protein-coupled receptor involved in the regulation of ovarian and testicular functions. In this study we demonstrate novel and unexpected patterns of receptor expression and regulation in fetal and adult rodent urogenital and adrenal tissues. Two rat LHR promoter fragments (approximately 2 and 4 kb) were shown to direct expression of the lacZ reporter in transgenic mice to gonads, adrenal glands, and kidneys, starting at 14.5 d post coitum, and to genital tubercles, starting at 11.5 d post coitum. These tissues were also found to express the full-length LHR mRNA and protein during rat fetal development, but, importantly, only immature receptors carrying unprocessed N-linked glycans were detected. After birth, the receptor gene activity ceased, except in the gonads, which started to express the mature receptor carrying fully processed N-linked glycans. Surprisingly, both LHR mRNA and mature protein levels were up-regulated substantially in pregnant female adrenal glands and kidneys at a time that coincides with differentiation of fetal urogenital tissues. Taken together, these results indicate that the LHR protein is expressed constitutively in gonadal and nongonadal urogenital tissues as well in adrenal glands, but its final functional maturation at the posttranslational level appears to be developmentally and physiologically regulated.

An alternative use of basic pGEX vectors for producing both N- and C-terminal fusion proteins for production and affinity purification of antibodies.

Glutathione S-transferase (GST) fusion proteins are widely used in protein production for pure immunogens, protein-protein, and DNA-protein interaction studies. Using basic pGEX vectors, foreign DNA is introduced to the C-terminus of the GST gene and the produced fusion proteins are C-terminally orientated. However, because the orientation of foreign polypeptides may have a very important role in the correct folding of the produced polypeptides, N-terminal fusion proteins are needed to express especially the N-terminus of the foreign polypeptide. Here, we introduce a novel use of the basic pGEX vectors for the production of N-terminal fusion proteins. In this procedure, PCR generated DNA fragments were cloned into the N-terminus of the GST gene in a unique EcoNI site located down-stream of the ATG initiation codon. The N-terminal fusion proteins were expressed in high quantities, easily solubilized, and affinity purified using our modification of current purification protocols. We also introduce here a new modification of the affinity purification of antibodies using covalently crosslinked GST and fusion proteins to glutathione-agarose beads. Our procedure was tested successfully for producing antibodies against both N- and C-terminus of the luteinizing hormone/chorionic gonadotropin receptor.

Identification and structural characterization of the neuronal luteinizing hormone receptor associated with sensory systems.

The luteinizing hormone receptor (LHR) is a G protein-coupled receptor involved in regulation of ovarian and testicular functions. Here we show that the receptor is present also in specific areas of the peripheral and central nervous system and may thus have a broader functional role than has been anticipated. Full-length LHR mRNA and two receptor protein species of M(r) 90,000 and 73,000, representing mature and precursor forms, respectively, were expressed in adult and developing rat nervous tissue, starting at fetal day 14.5. The receptor was capable of ligand binding because it was purified by ligand affinity chromatography, and human chorionic gonadotropin and LH were able to displace (125)I-labeled human chorionic gonadotropin binding to fetal head membranes in a dose-dependent manner. Finally, two 5'-flanking sequences ( approximately 2 and 4 kb) of the rat LHR gene were shown to direct expression of the lacZ reporter to specific areas of the peripheral and central nervous system in fetal and adult transgenic mice, especially to structures associated with sensory, memory, reproductive behavior, and autonomic functions. Importantly, the transgene activity was confined to neurons and colocalized with the cytochrome P450 side chain cleavage enzyme. Taken together, these results indicate that the neuronal LHR is a functional protein, implicating a role in neuronal development and function, possibly by means of regulating synthesis of neurosteroids.