Mariel Birnbaumer

Molecular cloning of the chicken progesterone receptor

To define the functional domains of the progesterone receptor required for gene regulation, complementary DNA (cDNA) clones encoding the chicken progesterone receptor have been isolated from a chicken oviduct lambda gt11 cDNA expression library. Positive clones expressed antigenic determinants that cross-reacted with six monospecific antibodies derived from two independent sources. A 36-amino acid peptide sequence obtained by microsequencing of purified progesterone receptor was encoded by nucleotide sequences in the longest cDNA clone. Analysis of the amino acid sequence of the progesterone receptor deduced from the cDNA clones revealed a cysteine-rich region that was homologous to a region found in the estrogen and glucocorticoid receptors and to the avian erythroblastosis virus gag-erb-A fusion protein. Northern blot analysis with chicken progesterone receptor cDNAs indicated the existence of at least three messenger RNA species. These messages were found only in oviduct and could be induced by estrogens.

Mutations and diseases of G protein coupled receptors.

Currently known disease-causing mutations in G protein coupled receptors are reviewed and discussed in conjunction with other naturally occurring receptor mutations. Special emphasis is made on opsin, vasopressin and MSH receptor mutations and what they tell are beginning to tell us about the inner workings of this superfamily of signalling molecules.

Phosphorylation of the V2 Vasopressin Receptor

The V2 vasopressin receptor undergoes ligand-induced sequestration and desensitization (Birnbaumer, M., Antaramian, A., Themmen, A. P. N., and Gilbert, S. (1992) J. Biol. Chem. 267, 11783-11788). The V2 receptor expressed in transfected cells labeled with [32P] orthophosphate was phosphorylated following the addition of 100 nM arginine vasopressin (AVP). Phosphorylation was complete 5 min after addition of AVP, and was not stimulated by increased levels of Ca2+ or cAMP. The half-maximal dose of AVP that stimulated phosphorylation was 2.4 ± 0.4 nM, similar to the receptor KD of 4.5 ± 0.4 nM. The role of phosphorylation on receptor desensitization was investigated by studying two vasopressin receptors 14 and 27 amino acids shorter than the wild type receptor. The missing segments were not needed for normal ligand binding or coupling to Gs, but the last 14 amino acids were required for phosphorylation. The truncated receptors exposed to 100 nM AVP were sequestered and desensitized. The R137H V2R mutant receptor that binds vasopressin with wild type-like affinity and does not couple to Gs (Rosenthal, W., Antaramian, A., Gilbert, S., and Birnbaumer, M. (1993) J. Biol. Chem. 268, 13030-13033) was phosphorylated and subjected to ligand-induced sequestration. These results established that phosphorylation is not essential for sequestration and desensitization of the V2 vasopressin receptor. Furthermore, they revealed that the conformation acquired after ligand occupancy is necessary for receptor phosphorylation and sequestration, while coupling to Gs is not.

Molecular cloning of a human gene (S31) encoding a novel serotonin receptor mediating inhibition of adenylyl cyclase

We report the molecular cloning of human gene (S31) containing an open reading frame of 1095 nucleotides, which encodes a protein of 365 amino acids. The encoded protein contains seven hydrophobic putative transmembrane domains considered the hallmark of G protein‐coupled receptors. The amino acid sequence shows highest homology to receptors for serotonin (5‐hydroxytryptamine). Expression of this receptor in murine Ltk − cells conferred upon these cells the ability to respond to serotonin by inhibition of adenylyl cyclase. No response was observed to isoproterenol, epinephrine, histamine, dopamine or melatonin in the transfected cells. We propose that the human gene S31 encodes a novel serotonin receptor.

Arrestin effects on internalization of vasopressin receptors

Arrestins have been shown to facilitate the recruitment of G protein-coupled receptors to the clathrin-coated vesicles that mediate their internalization. After (8)Arg-vasopressin-induced internalization, the human V2 vasopressin receptor failed to recycle to the cell surface, whereas the vasopressin type 1a receptor (V1a) subtype did. The possibility that the lack of recycling could identify a novel role for arrestins was investigated by examining the effect of coexpressing wild-type and dominant negative arrestins on the recycling of wild-type and mutant V2 and V1a receptors. Coexpression of the V1a or V2 receptors with the last 100 amino acids of arrestin reduced significantly their internalization, whereas coexpression of wild-type and mutant arrestins had diverse effects on internalization. Arrestin3 but not arrestin2 increased the internalization of the V1aR without altering its recycling pattern. Both nonvisual arrestins enhanced vasopressin type 2 receptor (V2R) internalization, inducing the appearance of a pool of recycling receptor in addition to the nonrecycling pool. The effect of arrestins on the internalization of the chimeric V1a/V2 receptor and its reciprocal chimera was specified by the identity of the carboxyl-terminal segment. The S363A mutation that confers recycling to the V2R did not alter its interaction with arrestins. Truncation of the carboxyl-terminal segment of the V2R impaired ligand-induced internalization that could be fully restored by wild-type arrestins. Internalization of the V2 and V1a receptors required dynamin GTPase activity.

Vasopressin Receptor Mutations and Nephrogenic Diabetes Insipidus

X-linked recessive nephrogenic diabetes insipidus is caused by mutations in the gene encoding the V2 vasopressin receptor (V2R), the mediator of the antidiuretic effect of arginine vasopressin (AVP) in mammalian kidneys. Upon binding to AVP, the receptor activates the G protein Gs, stimulating a phosphorylation cascade that promotes translocation of presynthesized water channels to the apical surface of the principal cells lining the last segments of the nephron. The presence of these channels allows the flow of water from the hypotonic lumen of the nephron into the hypertonic interstitium. More than 100 different mutations have been identified since the receptor gene was characterized--in most cases one per family, although some families bear two and three mutations in the same gene. The frequency of the de novo mutations identified suggests that the DNA at the end of the long arm of the X chromosome is very susceptible to alteration. The mutations are scattered within the coding region, not confined to a particular segment of the receptor protein, and in most cases confined to a single amino acid change that significantly reduces the number of receptors present on the plasma membrane. Some mutations do not affect protein synthesis but significantly reduce the coupling efficiency between the receptor and G protein. Analysis of the biochemical impact of the mutations has provided valuable information about the synthesis and regulation of the receptor.

Maturation of receptor proteins in eukaryotic expression systems.

Transient and stable expression in eukaryotic cells is commonly used to examine receptor function. Characterization of the V2 vasopressin receptor synthesized in transiently transfected cells revealed the presence of large quantities of immature protein and a small fraction of fully mature protein. The immature protein was characterized by its sensitivity to endoglycosidase H treatment, abnormal migration in SDS PAGE, and a tendency to form aggregates. Prevention of protein glycosylation by mutagenesis increased the fraction of mature protein produced, but did not eliminate the need for the maturation step. On the other hand, stably transfected cells produce almost exclusively mature receptor protein with a t1/2 of 6 h, while the immature form has a t1/2 of 20 min. In the absence of N-linked glycosylation the t1/2 of the mature V2 receptor in stably transfected cells was reduced to 4.5 h. In transient expression experiments the immature receptor proteins exhibited a prolonged t1/2 of about 8 h. Comparison of the half life of the immature form of the wild type and the R137H mutant V2 receptor did not reveal differences despite the lower amounts of mutant mature receptor detected by binding.

PHOSPHORYLATION AND RECYCLING KINETICS OF G PROTEIN-COUPLED RECEPTORS

The rate of ligand-induced phosphorylation of the V2 and V1a vasopressin receptors was characterized in HEK 293 cells. Both receptors were phosphorylated predominantly by GRKs, and the V1a receptor was also phosphorylated by protein kinase C regardless of the presence or absence of ligand. Phosphorylation of the V1aR catalyzed by GRKs reached maximal values at the shortest measured time: 15 seconds, and decayed rapidly with a t1/2 of 6 min in the continuous presence of AVP. In agreement with the hypothesis that dephosphorylation must precede receptor recycling to the cell surface, the V1aR returned rapidly to the cell surface after removal of the hormone from the medium. Phosphate incorporation into the V2R proceeded at a slower pace, and the internalized phosphorylated receptor failed to recycle to the cell surface and retained its phosphate for a long time in the presence or absence of ligand. A single mutation in the carboxy terminus of the V2R accelerated de-phosphorylation of the protein and conferred recycling properties to the V2R. These experiments provided molecular evidence for the hypothesis that internalization is required for de-phosphorylation and recycling of reactivated G protein coupled receptors to the cell surface.

The V2 vasopressin receptor mutations and fluid homeostasis

Although three different G-protein coupled receptors have been identified for arginine vasopressin, a significant physiological role has been recognized only for the V2 subtype that controls water homeostasis. Identification of the gene encoding the V2 vasopressin (or antidiuretic hormone) receptor enabled researchers to test the hypothesis that mutations of this gene were responsible for X-linked recessive nephrogenic diabetes insipidus. The affected patients are unable to concentrate their urine and as a consequence live in constant danger of dehydration that can cause death, particularly in infancy, or lead to severe hypernatremia that can impair their intellectual and physical development. The danger of severe dehydration diminishes in the adult patients, although they remain highly susceptible to this condition for the rest of their lives.

Molecular basis of regulation of ionic channels by G proteins

Publisher Summary This chapter presents primary structure of G proteins as deduced from purified proteins and cloned subunits. It also discusses their functions and presents data on direct regulation of ionic channels by G proteins. Experiments on expression of subunits, either in bacteria or by in vitro translation of messenger RNA (mRNA) synthesized from complimentary DNA (cDNA), are discussed as tools for definitive assignment of function to a given G protein. The chapter also discusses the dynamics of G protein-mediated signal transduction. The key points discussed in the chapter include the existence of two superimposed regulatory cycles in which G proteins dissociate into α plus βγ upon activation by guanosine triphosphate (GTP) and where the dissociated α subunits hydrolyze GTP. The chapter emphasizes the action of receptors to catalyze rather than regulate by allostery the activation of G proteins by GTP and also the role of subunit dissociation, without which receptors cannot act as catalysts. It also provides an overview on intramembrane networking of G protein-mediated receptor-effector coupling.