Ghsr Expression

The expression patterns of the type 1a and type 1b GHS-Rs were studied by ribonu-clease protection analysis in human and rat tissues and by in situ hybridization histochemistry in rhesus hypothalamus and rat brain and pituitary. Functional assessment of sucrose gradient-fractionated poly (A)+ mRNA from swine pituitary gave a single peak of GHS-R activity in the size range 1.6-2.3 kb (Fig. 7). However, attempts at detecting GHS-R mRNA by Northern blotting analysis have been unsuccessful, even though control mRNAs for other GPC-Rs could easily be detected. The authors attribute the difficulty in detecting GHS-R mRNA by Northern blotting analysis to its low abundance and potential size heterogeneity. TRH and GnRH receptors were also readily detected functionally. PCR amplification of the swine pituitary GHS-R cDNA sequences from among 11 pools of an unamplified pituitary cDNA library (110,000 individual cDNAs/pool) resulted in GHS-R cDNA identification in only 4 of 11 pools. Therefore, receptor cDNA abundance is most likely <1 in roughly 300,000. The more sensitive techniques of RNase protection and in situ hybridization proved more revealing. Table 4 summarizes the GHS-R expression data obtained from several species. GHS-R expression could be confirmed in multiple hypothalamic nuclei and the pituitary, as well as in other brain regions (e.g., hippocampus) and the pancreas. GHS-R transcripts have not been detected in numerous other tissues using RNase protection, including stomach, liver, heart, fetal brain, testis, thymus, adrenal gland, uterus, spinal cord, bone marrow, thyroid, and lung.

Fig. 6. Chromosomal location of the human and murine GHS-R gene. (A) Fluorescent in situ hybridization mapping for determination of the chromosomal location of the human GHS-R gene. Arrows highlight the location of the GHS-R gene. Arrowheads highlight the location of a chromosome 3 specific marker. A schematic Giemsa chromosome 3 banding pattern (ideogram) is shown on the right. The human GHS-R gene maps to chromosome 3Q26.2. (B) Fluorescent in situ hybridization mapping of the position of the mouse GHS-R gene. Arrows highlight the location of the GHS-R gene. Arrowheads highlight the location of a chromosome 3 specific marker. A schematic Giemsa mouse chromosome 3 banding pattern (ideogram) is shown on the right. The mouse GHS-R gene maps to chromosome 3A3.

Fig. 7. Determination of the approximate size of GHS-R mRNA by sucrose gradient fractionation of swine pituitary poly (A)+ mRNA. Swine poly (A)+ mRNA was size fractionated by sucrose gradient density fractionation. Aliquots from individual fractions were taken and tested in Xeno-pus oocytes for the presence of GHS-R mRNA. The histogram outlines the aequorin bioluminescence readout of GHS-R mRNA-derived signals (the ligand used to evoke a response was 1 \iM MK-0677). TRH and GnRH responses were also evaluated as controls.

Fig. 7. Determination of the approximate size of GHS-R mRNA by sucrose gradient fractionation of swine pituitary poly (A)+ mRNA. Swine poly (A)+ mRNA was size fractionated by sucrose gradient density fractionation. Aliquots from individual fractions were taken and tested in Xeno-pus oocytes for the presence of GHS-R mRNA. The histogram outlines the aequorin bioluminescence readout of GHS-R mRNA-derived signals (the ligand used to evoke a response was 1 \iM MK-0677). TRH and GnRH responses were also evaluated as controls.

Table 4

Summary of the Distribution of GHS-R Expression in Different Tissues^

Table 4

Summary of the Distribution of GHS-R Expression in Different Tissues^

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