Danijela Markovic

Differential responses of corticotropin-releasing hormone receptor type 1 variants to protein kinase C phosphorylation.

Corticotropin-releasing hormone (CRH) regulates diverse biological functions in mammals, through activation of two types of specific G protein-coupled receptors that are expressed as multiple mRNA spliced variants. In most cells, the type 1α CRH receptor (CRH-R1α) preferentially activates the Gs-adenylyl cyclase signaling cascade. CRH-R1α-mediated signaling activity is impaired by insertion of 29 amino acids in the first intracellular loop, a sequence modification that is characteristic of the human-specific CRH-R1β variant. In various tissues, CRH signaling events are regulated by protein kinase C (PKC). The CRH receptors contain multiple putative PKC phosphorylation sites that represent potential targets. To investigate this, we expressed recombinant CRH-R1α or CRH-R1β in human embryonic kidney 293 cells and analyzed signaling events after PKC activation. Agonist (oxytocin) or phorbol 12-myristate 13-acetate-induced activation of PKC led to phosphorylation of both CRH-R1 variants. However, CRH-R1α and CRH-R1β exhibited different functional responses to PKC-induced phosphorylation, with only the CRH-R1β susceptible to cAMP signaling desensitization. This was associated with a significant decrease of accessible CRH-R1β receptors expressed on the cell surface. Both CRH-R1 variants were susceptible to homologous desensitization and internalization following treatment with CRH; however, PKC activation increased internalization of CRH-R1β but not CRH-R1α in a β-arrestin-independent manner. Our findings indicate that CRH-R1α and -R1β exhibit differential responses to PKC-induced phosphorylation, and this might represent an important mechanism for functional regulation of CRH signaling in target cells.

Structural determinants critical for localization and signaling within the seventh transmembrane domain of the type 1 corticotropin releasing hormone receptor: lessons from the receptor variant R1d.

The type 1 CRH receptor (CRH-R1) plays a fundamental role in homeostatic adaptation to stressful stimuli. CRH-R1 gene activity is regulated through alternative splicing and generation of various CRH-R1 mRNA variants. One such variant is the CRH-R1d, which has 14 amino acids missing from the putative seventh transmembrane domain due to exon 13 deletion, a splicing event common to other members of the B1 family of G protein-coupled receptors. In this study, using overexpression of recombinant receptors in human embryonic kidney 293 and myometrial cells, we showed by confocal microscopy that in contrast to CRH-R1alpha, the R1d variant is primarily retained in the cytoplasm, although some cell membrane expression is also evident. Use of antibodies against the CRH-R1 C terminus in nonpermeabilized cells showed that membrane-expressed CRH-R1d contains an extracellular C terminus. Interestingly, treatment of CRH-R1d-expressing cells with CRH (100 nM) for 45-60 min elicited functional responses associated with a significant reduction of plasma membrane receptor expression, redistribution of intracellular receptors, and increased receptor degradation. Site-directed mutagenesis studies identified the cassette G356-F358 within transmembrane domain 7 as crucial for CRH-R1alpha stability to the plasma membrane because deletion of this cassette caused substantial intracellular localization of CRH-R1 alpha. Most importantly, coexpression studies between CRH-R1d and CRH-R2beta demonstrated that the CRH-R2beta could partially rescue CRH-R1d membrane expression, and this was associated with a significant attenuation of urocotrin II-induced cAMP production and ERK1/2 and p38MAPK activation, suggesting that CRH-R1d might specifically induce heterologous impairment of CRH-R2 signaling responses. This mechanism appears to involve accelerated CRH-R2beta endocytosis.

Identification of a Novel Corticotropin-Releasing Hormone Type 1β-Like Receptor Variant Lacking Exon 13 in Human Pregnant Myometrium Regulated by Estradiol-17β and Progesterone

Two types of CRH receptors mediate the diverse biological functions of CRH and CRH-related peptides. The type 1 CRH-R (CRH-R1) is extensively targeted by pre-mRNA splicing mechanisms that give rise to multiple mRNA splice variants. RT-PCR amplification of CRH-R1 sequences from human myometrium yielded cDNAs that encode a novel CRH-R1 splice variant with structural characteristics identical with CRH-R1β except a 14-amino acid deletion in the seventh transmembrane domain characteristic of the CRH-R1d. Transient expression of the hybrid CRH-R1 variant (CRH-R1β/d) in human embryonic kidney 293 cells revealed primarily intracellular expression, although some plasma membrane protein expression was also detectable. CRH bound to CRH-R1β/d with affinity comparable with the CRH-R1β; however, it was unable to stimulate adenylyl cyclase or other second messengers. Using a semiquantitative RT-PCR assay, CRH-R1β/d mRNA transcript was detected in human pregnant, but not nonpregnant, myometrium as early as 31 wk of gestation. Furthermore, in human pregnant myometrial cells, the relative expression of CRH-R1β and CRH-R1β/d mRNA appeared to be regulated by steroids; CRH-R1β/d mRNA expression was increased by estradiol-17β, whereas CRH-R1β mRNA levels were increased by progesterone. Progesterone also substantially increased CRH-R1α mRNA levels and cellular responsiveness to CRH as determined by increased agonist binding and cAMP production as well as resistance to CRH-R heterologous desensitization by phorbol esters. These results provide novel evidence for distinct patterns of CRH-R1 splicing and identify specific steroid-mediated regulation of CRH-R1 variant expression, which might be important for modulating CRH actions during human pregnancy and labour.

Estrogen Alters the Splicing of Type 1 Corticotropin-Releasing Hormone Receptor in Breast Cancer Cells

Altered splicing of a stress hormone receptor correlates with estrogen receptor–positive breast cancer. Altered Stress Hormone Receptor Splicing in Breast Cancer The stress hormone CRH (corticotropin-releasing hormone) is reported to both promote and inhibit breast cancer cell behavior. Lal et al. characterized kinase phosphorylation and transcriptional changes induced by CRH in an estrogen receptor–positive (ER+) breast cancer cell line, MCF7. The response to CRH was dynamic, with an early phase associated with phosphorylation of kinases associated with proliferation that was mediated by CRH receptor type 1 (CRH-R1), and a later phase associated with the phosphorylation of β-catenin and reduced migration of cultured MCF7. Estrogen prevented the early kinase response and the later inhibition of cell migration. Estrogen increased an alternatively spliced CRH-R1 transcript [CRH-R1(Δ12)] by decreasing the expression of the gene that encodes splicing factor SRp55. ER+ breast cancer tissue had decreased abundance of SRp55-encoding messenger RNA and increased abundance of CRH-R1(Δ12) compared with ER− breast cancers, suggesting that estrogen may disrupt the response of breast cancer cells to CRH by altering receptor splicing. Hormonal stress response is associated with the pathogenesis of disease, including cancer. The role of the stress hormone CRH (corticotropin-releasing hormone) in breast cancer is complex, and its abundance and biological activity may be modulated by estrogen. In the estrogen receptor–positive (ER+) malignant mammary epithelial cell line MCF7, CRH activated numerous kinases and downstream effectors, at least some of which were mediated by the CRH receptor type 1 (CRH-R1). CRH also increased the transcription of many genes that encode effectors, transcriptional targets, or regulators associated with estrogen signaling. Estrogen increased the abundance of the mRNA encoding CRH-R2 and an alternative splice variant encoding CRH-R1 in which exon 12 was deleted [CRH-R1(Δ12)]. Estrogen inhibited the expression SRSF6, which encodes serine/arginine-rich splicing factor 55 (SRp55). An increase in CRH-R1(Δ12), in response to either estrogen or SRp55 knockdown, dampened the cellular response to CRH and prevented its inhibitory effects on cell invasion. SRp55 knockdown also induced additional splicing events within exons 9 to 12 of CRH-R1, whereas overexpression of SRp55 prevented estrogen-induced generation of CRH-R1(Δ12). ER+ breast tumors had increased CRH-R2 and CRH-R1(Δ12) mRNA abundance, which was associated with decreased abundance of the mRNA encoding SRp55, compared with the amounts in ER− tumors, suggesting that estrogen contributes to the pathophysiology of ER+ breast cancer by altering CRH receptor diversity and disrupting CRH-mediated signaling.